Effects of Shenmai Injection on proliferation, migration, invasion and angiogenesis of colorectal carcinoma vascular endothelial cells

Abstract

Background:

Colorectal cancer, being 1 of the most significant malignant tumors globally, poses a substantial risk to human health. Unfortunately, its 5-year survival rate stands at a mere 65%. There remains an urgent need for the development of novel treatments to combat this detrimental malignancy effectively. The Shenmai Injection (SMI) is a Chinese medicine that has been proven to have significant clinical efficacy in the treatment of cardiovascular diseases. This study aimed to examine the impact of SMI on the proliferation, migration, invasion, and angiogenesis of tumor-derived endothelial cells (Td-EC).

Methods:

Human umbilical vein endothelial cells (HUVEC) induced Td-EC, and HUVEC were treated with conditioned media from the human colorectal carcinoma cells (HCT116). The effects of HCT116 on the proliferation, migration, and invasion of hepatocellular carcinoma cells after treatment of SMI were observed by MTS assay and Transwell techniques. Additionally, an angiogenesis experiment was used to investigate Td-EC tube formation capacity.

Results:

SMI had a significant inhibiting effect on the proliferation, migration, and invasion of HCT116. SMI was also able to inhibit the angiogenesis of Td-EC. Notably, SMI did not have any effect on the normal endothelium.

Conclusion:

SMI has obvious antiproliferation, migration, infiltration, and neogenesis effects on HCT116.

1. Introduction

As the third most common cancer in the world, colorectal cancer (CRC) still lacks effective medical intervention and an adequate understanding of its pathogenesis.^[1] Epidemiological data reveal that CRC has a mortality rate of 9.4%, making it the second deadliest cancer, and its development poses a significant threat to the lives of patients.^[2] The ideal treatment for CRC is to tumor remove the tumor through surgical procedures completely. However, nearly a quarter of CRCs are diagnosed with metastasis.^[3] For unresectable metastatic CRC, treatments include cytotoxic chemotherapy, biological therapy, and immunotherapy.^[4] There are also successful first-line drugs like the anti-EGFR agent cetuximab and the antiangiogenesis agent bevacizumab.^[5] However, the median overall survival time for patients on first-line chemotherapy is only 30 months.^[6] Furthermore, patients experience significant discomfort due to adverse effects. Hence, it is crucial to investigate new pharmaceutical compounds as potential candidates for targeting this malignancy.

Tumor cells exhibit the characteristics of rapid growth and a vigorous metabolism, which require a substantial supply of nutrients. In the early stages of tumorigenesis, cells can access nutrients directly from their surroundings, but at more than 2 mm, new blood vessels are needed to provide nutrients.^[7,8] Angiogenesis has been proven to be 1 of the hallmarks of cancer and is involved in CRC progression and metastasis. Antiangiogenic drugs have emerged as a promising therapeutic approach for the treatment of CRC. In first-line chemotherapy, combined use of Bevacizumab can increase patients’ disease-free development survival time to 12 months.^[9] In the context of second-line therapy, antiangiogenic drugs have been observed to improve overall survival rates. In addition, although immunotherapy brings hope to many patients with advanced colorectal cancer, the efficacy of immunotherapy is still limited for patients with microsatellite instability-high (MSI-H): DNA mismatch repair-deficient (dMMR) tumors.^[10] For microsatellite stable (MSS) tumors, immunotherapy is not beneficial. However, antiangiogenic drugs have the function of promoting the normalization of tumor blood vessels. Therefore, their combination with immunotherapy is very promising. Despite this potential, current antiangiogenic drugs are associated with significant side effects, including arterial hypertension, arterial thromboembolism, congestive heart failure, etc. Finding efficient antiangiogenic medications has consequently become a pressing issue that has to be resolved in certain extreme circumstances, they may even be life-threatening.

Traditional Chinese medicine (TCM) has accumulated abundant data on clinical syndrome and drug use and plays a crucial role in clinical diagnosis and treatment.^[11] As a TCM, Shenmai Injection (SMI) is an effective ingredient separated from ginseng and Ophiopogon japonicas.^[12] It is widely used in clinics and can effectively prevent and treat myocardial injury caused by doxorubicin.^[13] Heart failure is the common outcome of a variety of cardiovascular and cerebrovascular diseases. The primary determinant for the advancement and decline of heart failure is the atypical energy metabolism shown by the cardiac muscle. TCM has some common characteristics in preventing and treating heart failure and improving myocardial energy balance.^[14] SMI can reduce myocardial injury caused by adriamycin by regulating the expression of inflammatory factors. SMI has also been reported to have a supportive role in tumor immunotherapies^[15,16] In short, the regulatory effect of SMI on blood vessels has been broadly reported. However, its role in tumor vessels is not clear.

In this study, we aimed to perform a pilot exploration to assess the antiangiogenesis property of the SMI in the tumor environment.

2. Materials and Methods

2.1. Drug

Zhengda Qingchunbao Pharmaceutical (Hangzhou, China) purchased SMI. China National Drug Administration (CNDA) approval number: Z33020021. The active components in SMI are red ginseng (0.1 g/mL) and dwarf lilyturf tuber (0.1 g/mL).

2.2. Cell culture

Human colorectal carcinoma cells (HCT116) and the human umbilical vein endothelial cells (HUVEC) were obtained from Zhejiang Meisen Cell Technology (Zhejiang, China). HCT116 were cultured with 10% FBS, while HUVEC were cultured with 5% FBS and 10 ng/L of vascular endothelial growth factor (VEGF) in a humidified incubator at 37 °C with 5% CO₂.

The Td-EC and intact medium, which contained 50% HCT116, were induced in HUVEC for a duration of 48 hours. Td-EC was detected using a transmission electron microscope, revealing its association with distinct tumor neovascularization. This characteristic enables its differentiation from the regular endothelium.^[17] Consequently, we selected TEM1 and TEM8 as the test subjects for the Td-EC experiment.

3. MTS assay

The viability of HCT116 were recorded post-SMI treatment using the MTS assay. In detail, HCT116 during the logarithmic growth phase were harvested and plated at a density of $1\times {10}^5$ cells/well in a 96-well plate, with 180 µL of medium per well. Then, the SMI was prepared at several concentrations viz 0, 8, 10, 12, 14, and 16 μL and inserted after cell adherence to each well separately. The treatment lasted for 0, 24, and 48 hours. After the specified incubation periods, MTS solution (Life-iLab Biotech, Shanghai, China) was added to each well and allowed to react for 1 hour. During this incubation, the 96-well plate was placed in the incubator to maintain optimal conditions. Finally, the optical density was measured at a wavelength of 490 nm to determine the absorption values, which indicated cell viability.

4. Quantitative polymerase chain reaction

TRIzol (Life-iLab Biotech) was used to separate HUVEC and Td-EC from RNAs. Using the universal cDNA Synthesis Kit II for cDNA Synthesis. The relative expression of SYBR Green I (Life-iLab Biotech) was determined by SYBR HotStart Taq DNA polymerase and method. The following are the primers used in this experiment:

$${\displaystyle \begin{array}{l}{\displaystyle TEM{15}^{\prime }-CTGATGGGTGAGGTCTGGTT-3^{\prime }}\end{array}}$$

$${\displaystyle \begin{array}{l}{\displaystyle TEM{85}^{\prime }-GTCTCCTCCTGGCAGAACTT-3^{\prime }}\end{array}}$$

$${\displaystyle \begin{array}{l}{\displaystyle \beta -actin{5}^{\prime }-CACAGAGCCTCGCCTTTGC-3^{\prime }}\end{array}}$$

$${\displaystyle \begin{array}{l}{\displaystyle VEGF-qF{15}^{\prime }-TTGCCTTGCTGCTCTACCTCCA-3^{\prime }}\end{array}}$$

$${\displaystyle \begin{array}{l}{\displaystyle VEGF-qR{15}^{\prime }-GATGGCAGTAGCTGCGCTGATA-3^{\prime }}\end{array}}$$

$${\displaystyle \begin{array}{l}{\displaystyle PDGFB-qF{15}^{\prime }-GAGATGCTGAGTGACCACTCGA-3^{\prime }}\end{array}}$$

$${\displaystyle \begin{array}{l}{\displaystyle PDGFB-qR{15}^{\prime }-GTCATGTTCAGGTCCAACTCGG-3^{\prime }}\end{array}}$$

$${\displaystyle \begin{array}{l}{\displaystyle TGF-\beta -qF{15}^{\prime }-TACCTGAACCCGTGTTGCTCTC-3^{\prime }}\end{array}}$$

$${\displaystyle \begin{array}{l}{\displaystyle TGF-\beta -qR{15}^{\prime }-GTTGCTGAGGTATCGCCAGGAA-3^{\prime }}\end{array}}$$

$${\displaystyle \begin{array}{l}{\displaystyle FGFb-qF{15}^{\prime }-AGCGGCTGTACTGCAAAAACGG-3^{\prime }}\end{array}}$$

$${\displaystyle \begin{array}{l}{\displaystyle FGFb-qR{15}^{\prime }-CCTTTGATAGACACAACTCCTCTC-3^{\prime }}\end{array}}$$

$${\displaystyle \begin{array}{l}{\displaystyle Ang2-qF{15}^{\prime }-ATTCAGCGACGTGAGGATGGCA-3^{\prime }}\end{array}}$$

$${\displaystyle \begin{array}{l}{\displaystyle Ang2-qR{15}^{\prime }-GCACATAGCGTTGCTGATTAGTC-3^{\prime }}\end{array}}$$

$${\displaystyle \begin{array}{l}{\displaystyle Ang1-qF{15}^{\prime }-CAACAGTGTCCTTCAGAAGCAGC-3^{\prime }}\end{array}}$$

$${\displaystyle \begin{array}{l}{\displaystyle Ang1-qR{15}^{\prime }-CCAGCTTGATATACATCTGCACAG-3^{\prime }}\end{array}}$$

$${\displaystyle \begin{array}{l}{\displaystyle SNAIL-qF{15}^{\prime }-TCGGAAGCCTAACTACAGCGA-3^{\prime }}\end{array}}$$

$${\displaystyle \begin{array}{l}{\displaystyle SNAIL-qR{15}^{\prime }-AGATGAGCATTGGCAGCGAG-3^{\prime }}\end{array}}$$

5. Cell migration assay

When HUVEC and Td-EC ($4\times {10}^5$ cells/well) were implanted into 6-well plates, and the fusion rate was 90%. A single cell was manually scraped from the top of the p1000 straw to form a wound. Then, the cells were treated with 0, 8, and 16 μmol/L SMI for 48 hours. Following treatment, the cells were washed once with 1 mL of Dulbecco’s Modified Eagle Medium and then replaced with 2 mL of fresh Dulbecco’s Modified Eagle Medium. The culture was carried out at 37 °C, 5% CO₂ concentration, and moist conditions. After 48 hours, the cells were observed with a hand-held microscope and a digital camera (Olympus IX73, Olympus Corporation, Tokyo, Japan).

6. Cell apoptosis test

Apoptosis assay (Life-iLab Biotech) was conducted according to the instructions. Briefly, once the cells had reached 70% to 80% confluence, they were plated on a 6-well culture plate and then cultured for 24 hours at 37 °C with different dosages of SMI or vehicle control. The cells were then evaluated. Following the guidelines provided by the manufacturer, the cells were collected after 24 hours of incubation in SMI and proceeded by washing twice in PBS. Afterwards, dilute 10x Binding Buffer into 1x Binding Buffer with deionized water. Collect cells after trypsin digestion without EDTA at room temperature. The staining was performed according to the instructions, and analysis was performed using a flow cytometer (Cytoflex, Beckman Coulter, CA) Transwell invasion assay.

7. Transwell invasion assay

Cells were cultured in a serum-free medium (10 g/L BSA instead of serum) for 12 hours, followed by digestion and adjustment to a density of $2\times {10}^6$ cells/ mL. For cell inoculation, 100 µL of the cell suspension was added to the upper chamber (also containing 10 g/L BSA instead of serum), while 600 µL of 1% FBS was added to the lower chamber to avoid bubbles, with the cells cultured for 16 hours. For results statistics, the chamber was removed and carefully rinsed in PBS. The organelles on the film’s surface were carefully cleaned with yarn strips and placed in a 24-well plate. The organelles were then fixed using 4% paraformaldehyde for 20 minutes. After being treated with 0.1% crystal violet solution for 15 minutes, followed by 3 washes with PBS. The fixed samples were photographed under an inverted microscope. For statistical analysis, 3 random fields of view were counted per sample using a 10× objective lens, and the average cell count per field was calculated.

7.1. In vitro angiogenesis experiment

HUVEC and Td-EC were digested and adjusted to a concentration of $8.0\times {10}^6$ cells/ mL. The serum-free medium and Matrigel were precooled to prepare the basement membrane extract (BME). The BME was incubated at 37 °C for 30 minutes to allow it to solidify. A total of 300 μL of medium mixed with 10 μL of the cell suspension was carefully added on top of the solidified BME. After 24 hours of HUVEC seeding, treatment with various SMI concentrations or/or vehicle control was initiated. After the treatment was over, cells were washed, and the test tube was photographed with a phase-contrast microscope (Olympus IX73) of tube formation every 4 hours.

8. Statistical analysis

Each group of tests was repeated at least 3 times, and the results were calculated as the mean ± standard deviation. The Student-tests were used to compare the mean values to assess the significant differences between the 2 groups. P < .05 indicates that there is a considerable difference. All data processing was statistically analyzed by SPSS13.0. (SPSS, Chicago).

9. Results

9.1. SMI inhibits cell proliferation of HCT116

Using the MTS assay, the effects of SMI were analyzed to see how it affected the cell proliferation of the HCT116. The SMI was made in a variety of concentrations viz 0, 8, 10, 12, 14, and 16 μL, and was observed at 0, 24, and 48 hours. We found that SMI has a significant concentration- and time-dependent effect on the growth of hepatoma HCT116. SMI at concentrations of 14 and 16 microliters substantially reduced HCT116 growth, with the latter effect being more pronounced on day 2 (Fig. 1A). As a consequence of this, the results of the MTS assay demonstrated that SMI had a significant impact on inhibiting the growth of the HCT116.

Figure 1.

Wound healing assay: after 0, 24, and 48 hours of co-culture with different concentrations of SMI the wound scratched in each case was visualized. The results indicated that the SMI significantly inhibited the migration of HCT116 compared with untreated cells. SMI = Shenmai Injection.

9.2. SMI inhibits cell migration in HCT116

We implemented the Transwell invasion assay to demonstrate the molecular mechanism of SMI regulating tumor cell infiltration in HCT116. As shown in Fig. 1B and C, the migration of HCT116 decreased significantly. Likewise, the effect on cell movement was observed by wound healing tests. The results showcased that the SMI treatment induced migration inhibitory effects as was indicated by the wound closure after 24 hours (Fig. 3). Based on these findings and the outcomes of Transwell migration and wound healing tests, it was concluded that treating HCT116 with SMI greatly decreased their capacity to move in concentration- and time-dependent patterns.

Figure 3.

The apoptosis analysis: (A) HDT116 cells were treated with 0 and 16 μL of SMI for 24 hours showing dose-dependent apoptosis induction in dot-plot quadrants. (B) The percentage of apoptotic cells is presented as a bar graph. *P < .05. SMI = Shenmai Injection.

10. Td-EC reasoning and testing

We used polymerase chain reaction to detect TEM1 and TEM8 and looked into whether or not HUVEC might be transformed into Td-EC when exposed to the HCT116-conditioned medium. As shown in Figure 2A, TEM1 and TEM8 were not expressed in HUVEC but were significantly expressed in Td-EC. At the same time, HUVEC and Td-EC and their respective dosing groups were tested to explore the mechanisms of the effects of the drugs (Fig. 2B). The expression of Ang-1, Ang-2, fibroblast growth factor beta, platelet-derived growth factor B (PDGFB), and transforming growth factor β was significantly decreased, while the SNAIL gene and VEGF were increased. It indicated the induction of the Td-EC in the HCT116.

Figure 2.

Relative mRNA expression of HUVEC and Td-EC. Notes: The contents of TEM1 and TEM8 (A) were determined by PCR. Human umbilical cord vascular endothelial cells and Td-EC (B) were cultured in vitro with 16 uL SMI. Then Ang-1, Ang-2, FGFb, PDGFB, SNAIL, TGF-β, and VEGF were also detected by PCR. ****P < .0001. FGFb = fibroblast growth factor beta, HUVEC = human umbilical vein endothelial cells, PCR = polymerase chain reaction, PDGFB = platelet-derived growth factor B, SMI = Shenmai Injection, Td-EC = tumor-derived endothelial cells, TGF-β = transforming growth factor β, VEGF = vascular endothelial growth factor.

11. SMI induces apoptosis in HCT116

The annexin-V method was used to observe the apoptosis after 24 hours of the treatment with SIM at 0 and 16 μL. The assay results revealed that the SMI-treated cells showed the arrival of apoptosis compared to the untreated cells. The percentage of apoptotic cells increased remarkably on increasing the concentrations of the SMI, thus pointing out at dose dependence of apoptosis induction in HCT116 by SMI (Fig. 3A and B).

11.1. SMI inhibits Td-EC angiogenesis in vitro

Here, using fluorescence microscopy, we compared the effects of various dosages of SMI on angiogenesis and found that Td-EC is superior to HUVEC in its capacity to produce vascular tubes. SMI significantly inhibited the vascular formation of Td-EC. However, it had no significant effect on HUVEC (Fig. 4A and B). These results indicate that SMI treatment significantly inhibited tube formation and, consequently, angiogenesis in HCT116.

Figure 4.

Effects of SMI on Td-EC tube formation in vitro. Notes: HUVEC (A, B) and Td-EC (C, D) were treated with 16 μL SMI. DMEM was used as the negative control group, and the differences among groups were observed. The magnification is a hundred times. The scale bar is 100 μm. The tube formation number (%) is quantified in (E). *P < .05. DMEM = Dulbecco’s Modified Eagle Medium, HUVEC = human umbilical vein endothelial cells, SMI = Shenmai Injection, Td-EC = tumor-derived endothelial cells.

12. Discussion

Over 900,000 people die every year from colorectal cancer (CRC), making it 1 of the deadliest forms of cancer.^[18] Standard treatments for colorectal cancer include surgery and adjuvant chemotherapy. The malignant tumors that define cancer often recur or metastasize to other parts of the body. In general, in the process of cancer occurrence and development, the probability of recurrence and metastasis of early tumors is relatively low. In contrast, the probability with advanced stages will be significantly increased. As a traditional technology in China, TCM has received extensive attention in recent years. Traditional medicine is more regarded as a medical system with prevention and treatment functions than Western medicine. Especially in the field of Chinese herbal medicine, it has attracted more and more interest. There is no doubt that many medicines are derived from TCM and natural products. This paper highlights that SMI, a traditional Chinese herbal medicine for preventing and treating cardiovascular disease (CVD) in China, has potential as a key active antitumor component.^[19] However, research on the effects of SMI on CRC is still limited.

The occurrence and development of tumors depend on reprogramming cancer cell metabolism. Cancer cells use a variety of metabolic pathways to regulate their metabolic flow, allowing them meet their energy and substance requirements while also decreasing their sensitivity to oxidative stress.^[20] The regulation of fluxes was governed by cancer mutations and the nutritional factors derived from the surrounding environment.

Tumor microenvironment means the internal and external environment of the tumor. This term involves tissue structure, function, metabolism, and its internal microenvironment (nucleus and cytoplasm). Therefore, it is of equal importance to study and understand the tumor microenvironment and pay attention to the cancer cells themselves.^[21]

Normal cells that are co-cultured with cancer cells are known to be genetically stable, making them easier to target than genetically unstable cancer cells. That is 1 of the reasons why we chose Td-EC as the experimental object. Over the past few years, however, the vast complexity of these cells has become apparent. If 1 simply classifies the role of these cells as 1, such a conclusion appears inaccurate. Considering the process of cancer development as well as the organ where cancer appears, the cells in the tumor microenvironment are supposed to be either tumor suppressors or tumor supporters.^[22] Even the type of cancer, the development of the cells themselves, and the environment in which they grow can be factors.

The drug we selected functions to inhibit tumors by targeting the tumor microenvironment. SMI is widely used in clinical practice, as it has been shown to offer myocardial protection through network pharmacology, effectively achieving synergy across multiple targets and pathways.^[23]

Some signaling pathways are a key part of tumor angiogenesis. Ang-1 and Ang-2 are the hotspots in researching and developing antineoplastic drugs. In addition, Ang-1 is a crucial angiogenic molecule and plays a key role in the migration, adhesion, and survival of ECs. Ang-2 affects vascular degeneration by disrupting connections between endothelial cells and perivascular cells, promoting cell death. The promotion of angiogenesis is synergistically facilitated by Ang-2 and VEGF. Angiopoietins 1 and 2, along with VEGF, play a crucial role in the process of angiogenesis that occurs during tumor development. The down-regulation of these factors can have a significant impact on the advancement and growth of tumors. They are crucial in regulating angiogenesis, making many drugs regarded as angiopoietin. The signaling pathway is an important target. Fibroblasts promote the immunosuppressive tumor microenvironment of CRC metastasis by modulating immune cell phenotypes.^[24] Down-regulation of related factors such as fibroblast growth factor beta can cancel this immunosuppressive effect. PDGFB is vital in recruiting pericytes into blood vessels. Once PDGFB is lost, tumor vascular function and pericyte coverage are significantly impaired.^[25] In addition, the SNAIL gene has been found to accelerate the transformation of epithelial tumors, and the transformed cells will go through a series of processes, thus increasing the infiltration of M2 macrophages and the metastasis of tumor cells.^[26]

In this study, we selected Td-EC, a cell type that regulates the diameter and elasticity of blood vessels, closely associated with tumor growth and metastasis.^[27] The formation and progression of the tumor are intricately linked to the process of neovascularization. Metastasis and angiogenesis are fundamental attributes that define the malignant behavior exhibited by cancer cells. Metastasis is the cause of death that accelerates the progression of CRC.^[28] Angiogenesis serves as a key link in tumor progression. Tumor cells depend on angiogenesis because oxygen and nutrients are essential for tumor cells to proliferate and expand.^[29] Consequently, targeting tumor angiogenesis has emerged as a promising strategy in anticancer therapies. Tumor endothelial cells and common endothelial cells have many different characteristics. There is also more than 1 mechanism of tumor angiogenesis. Some tumor cells can mimic endothelial formation to form loops or networks similar to vascular systems. These loops or networks do not contain endothelial cells but do contain blood cells.^[30] Therefore, antitumor angiogenesis has become a very valuable antitumor drug. In addition, tumor-derived growth factors, such as VEGF, if not controlled by angiogenesis inhibitors, stimulate rapid angiogenesis, causing endothelial cells to produce loose blood vessels.^[31] VEGFR1 and VEGFR2 are more highly expressed in Td-EC than in normal endothelial cells. This shows that Td-EC will be more active in VEGF, which leads to accelerated angiogenesis in Td-EC and may improve its survival ability in a serum-free environment, which is very different from normal endothelial cells.^[32] Ang-2 can inhibit the binding of Ang-1 to Tie2 on Td-EC.^[33] This causes pericytes to detach and increases the vascular permeability of the tumor. The unique properties of Td-EC make it a target for specific drugs.^[34] TEM1 and TEM8 are unique to tumors and do not exist in physiological angiogenesis. Therefore, we choose TEM1 and TEM8 as the test objects of Td-EC.

The current study has certain limitations. Firstly, the presented data is generated from in vitro models, considering this is a pilot study. TCM inherently faces challenges, such as difficulties in quantification and variability in batches during drug preparation. Still, our work provides a new scenario for developing anticancer treatments. We will continue to explore the detailed mechanism in future research.

13. Conclusion

In summary, this study provides in vitro evidence of the antiproliferative effects of SMI injection on HCT116 and the antiangiogenesis property on tumor-derived vascular endothelial cells. Our study showed the potential of SMI in developing novel treatments for CRC.

Author contributions

Conceptualization: Xiangyu Liu.

Data curation: Xiangyu Liu.

Formal analysis: Xiangyu Liu.

Investigation: Xiangyu Liu.

Writing – original draft: Xiangyu Liu.

Writing – review & editing: Xiangyu Liu.