Breviscapine regulates the proliferation, migration, invasion, and apoptosis of colorectal cancer cells via the PI3K/AKT pathway

Colorectal cancer (CRC) is ranked as one of the most common malignancies with a high death rate. It has been discovered that breviscapine can alter the progression and development of various cancers. Nevertheless, the function and mechanisms of breviscapine in CRC progression have not yet been described. The cell proliferation capacity of HCT116 and SW480 cells was assessed using the CCK-8 and EdU assays. Cell apoptosis was tested through flow cytometry, and cell migration and invasion were examined using the transwell assay. Moreover, protein expression was examined through a western blot. Tumor weight and volume were assessed using the nude mice in vivo assay, and the Ki-67 protein expression was verified through the IHC assay. This study discovered that an increased dose of breviscapine (0, 12.5, 25, 50, 100, 200, and 400 μM) gradually reduced cell proliferation and increased apoptosis in CRC. Additionally, breviscapine restricted the migration and invasion CRC cells. Moreover, it was revealed that breviscapine inactivated the PI3K/AKT pathway and inhibited CRC progression. Finally, an in vivo assay demonstrated that breviscapine restrained tumor growth in vivo. It affected the CRC cells’ proliferation, migration, invasion, and apoptosis through the PI3K/AKT pathway. This discovery may offer new insights into CRC treatment.

. In addition, it up-regulates miR-7 to restrain the tumorigenesis of non-small cell lung cancer 18 , and it exhibits antitumor activity in hepatocellular carcinomas 19 . However, the effects of breviscapine on CRC progression have remained unclear. In this study, the role of breviscapine in CRC progression was investigated in detail. The findings indicated that breviscapine reduces cell proliferation, migration, and invasion and influences apoptosis in CRC by modulating the PI3K/AKT signaling pathway. This discovery offers a promising therapeutic agent that could ameliorate the treatment of patients with CRC.
Flow cytometry. The Annexin V-FITC Apoptosis Detection Kit (Beyotime, Shanghai, China) was utilized in measuring apoptosis. First, the HCT116 and SW480 cells (1 × 10 3 cells/well) were placed in 24-well plates and cultivated for 48 h. Then, the cells were harvested and resuspended in the binding buffer (100 µL), including Annexin V-FITC (5 µL) and PI (propidiumiodide) (5 µL), and incubated in the dark for 15 min. Finally, apoptosis was analyzed using flow cytometry (BD Biosciences, Franklin lakes, NJ, USA).
Wound healing assay. HCT116 and SW480 cells were grown to 80-90% confluence in six-well plates.
Then, cell scratching was performed using a sterile pipette tip (10 µL). The scratch distance was observed under a microscope at 0 h and after 24 h.
Nude mice in vivo assay. The male BALB/c nude mice (4-6 weeks old, n = 12) were procured from the Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). HCT116 cells (5 × 10 6 ) were injected subcutaneously into the flank region of the nude mice. When the tumors reached approximately 5 mm in diameter, the intraperitoneal administration of breviscapine (40 mg/kg) or 5-FU (25 mg/kg) was performed 22 . The control group was treated with an equal dose of DMSO. After 2 weeks, the mice were killed using an intraperitoneal injection with an overdose of pentobarbital. Importantly, all the animal experiments were approved by the Ethics Committee of Beijing Viewsolid Biotechnology Co. LTD (VS212601467) and were performed per the relevant guidelines and regulations. Furthermore, this study is reported in accordance with the ARRIVE guidelines (https:// arriv eguid elines. org). www.nature.com/scientificreports/ IHC assay. After dewaxing with ethanol, the mice tumors' tissue sections (4 μM) were mixed with H 2 O 2 in methanol. Then, the sections were mixed with the primary antibody Ki-67 (ab15580, 1 µg/mL, Abcam) overnight at 4℃. After washing, secondary antibodies (ab6721, 1:1000, Abcam) were added and further incubated. Next, the sections were dyed with diaminobenzidine (DAB) and redyed with hematoxylin. Lastly, the images were observed using fluorescence microscopy (IX-51, Olympus).

Statistical analysis.
The data obtained from this study were presented as the mean ± standard deviation (SD). SPSS 20.0 software (SPSS Inc., Chicago, IL, USA) was used for statistical analysis and presented using GraphPad Prism Software 8.0. In addition, the experimental procedures were repeated at least three times. Finally, any observed group variation was presented using the Student's t-test or a one-way analysis of variance (ANOVA) with the Tukey-Kramer multiple comparisons test. The results were considered statistically significant when p < 0.05.
Ethical approval. All animal experiments were approved by the Ethics Committee of Beijing Viewsolid Biotechnology Co. LTD (VS212601467) and all experiments were performed per relevant guidelines and regulations. Furthermore, the study is reported per the ARRIVE guidelines (https:// arriv eguid elines. org).

Results
Breviscapine regulated the proliferation and apoptosis of CRC cells. Breviscapine has been investigated regarding the development of many cancers, but its function in CRC is unclear. As displayed in Fig. 1A, the cell proliferation of HCT116 and SW480 was inhibited through breviscapine treatment using a dose-increasing technique, and breviscapine and 5-FU treatments both impaired cell viability (Fig. 1B). Further experiments using breviscapine (0, 25, 50, and 100 μM) were conducted in this study. Additionally, an EdU assay was performed to assess cell proliferation, and it was discovered that the EdU-positive cells (indicating cell proliferation ability) decreased with an increase in breviscapine concentration (0, 25, 50, and 100 μM) (Fig. 1C). During flow cytometry, the lower left quadrant contained living cells, the upper left quadrant had dead cells, the upper right quadrant had late apoptotic cells, and the lower right quadrant contained early apoptotic cells. The rate of apoptosis (the upper right quadrant + the lower right quadrant) increased with an increase in the breviscapine concentration (Fig. 1D). In addition, the expression of the PCNA protein decreased, while cleaved-caspase three and p53 expressions increased with an increase in breviscapine concentration (Fig. 1E). Altogether, breviscapine restrained cell proliferation and accelerated apoptosis in CRC.

Breviscapine attenuated the migration and invasion abilities of CRC cells in Transwell assays.
Next, through Transwell assays, it was demonstrated that cell migration and invasion were restricted with an increase in breviscapine concentration ( Fig. 2A,B).

Breviscapine inhibited cell migration and EMT process in CRC cells.
During the wound-healing assay, cell migration decreased after breviscapine treatment (Fig. 3A). At the same time, the EMT progress markers (the epithelial marker-E-cadherin and the mesenchymal marker-N-cadherin) were examined. The expression of N-cadherin was down-regulated, and E-cadherin expression was up-regulated with the increase in breviscapine concentration, indicating that the EMT process was restricted after breviscapine treatment (Fig. 3B). These results revealed that breviscapine limited the migration and invasion of CRC cells.
Breviscapine regulated cell proliferation and apoptosis through affecting the PI3K/AKT pathway. The PI3K/AKT pathway has been revealed to be a key regulator in CRC progression. Thus, breviscapine regulation of the PI3K/AKT pathway in CRC progression was investigated. The levels of p-PI3K and p-AKT decreased with the increase in breviscapine concentrations (Fig. 4A), while PI3K and AKT levels did not change. Moreover, it was discovered that the decreased p-PI3K and p-AKT levels facilitated by breviscapine (100 μM) were recovered after treatment with the PI3K/AKT activator (740Y-P) (Fig. 4B), and the reduced cell proliferation triggered by breviscapine was offset after 740Y-P treatment (Fig. 4C). In addition, apoptosis increased with breviscapine treatment; however, this change can be reversed after adding 740Y-P (Fig. 4D).

Breviscapine suppressed cell migration, invasion and EMT progress through affecting the PI3K/AKT pathway.
Furthermore, the decreased cell migration and invasion induced by the breviscapine treatment can be countered by adding 740Y-P ( Fig. 5A-C). Similarly, the down-regulated N-cadherin expression and the up-regulated E-cadherin expression induced by the breviscapine treatment can be neutralized after treatment with 740Y-P (Fig. 5D). In conclusion, breviscapine inactivated the PI3K/AKT pathway and inhibited CRC progression.

Breviscapine inhibited tumor growth in vivo.
Further investigation in vivo indicated that the tumor weight and volume decreased after breviscapine (40 mg/kg) treatment (Fig. 6A,B). In addition, the IHC assay revealed that protein expression of the cell proliferation marker, Ki-67, was lower in the breviscapine 40 mg/kg group (Fig. 6C). These findings indicated that breviscapine inhibits tumor growth in vivo.

Discussion
Due to the advancements in phytochemistry, multiple nontoxic and effective components have been isolated from plants, which offer a broad alternative resource base for developing novel antitumor drugs 23,24 . For example, breviscapine derived from E. breviscapus is one such natural flavonoid. Several studies have demonstrated that breviscapine has various pharmacological applications. For instance, it offers anti-inflammatory, renal, cardiovascular, and neural protection. Moreover, breviscapine regulates the PI3K/Akt/GSK-3β pathway and suppresses inflammation and myocardial apoptosis after coronary microembolization 25 . In addition, it exhibits antioxidant and anti-inflammatory properties that improve cognitive impairments triggered by transient cerebral ischemia and reperfusion 26 . Furthermore, breviscapine affects the Nrf2 signaling pathway and modulates IL-6 expression, thereby displaying neuroprotective effects and relieving traumatic brain injury 27,28 . www.nature.com/scientificreports/ It has generally been recognized that breviscapine has antitumor properties [15][16][17][18][19] ; however, its function in CRC remains unclear. Compared to previous research, it was discovered in this study that breviscapine regulates the proliferation and apoptosis of CRC cells and restricts tumor growth in vivo.
Cancer metastasis is a complex process in which tumor cells break away from the primary tumor, settle, and growth in other locations in the body 29,30 . Whether early or late, many cancer patients may eventually become metastatic 31 . Unfortunately, the current treatment methods have difficulty curing the metastatic lesions of distant organs, which results in the death of about 90% of cancer patients 32,33 . Thus, tumor metastasis is the principal challenge regarding cancer-related death 33 . Therefore, exploring the mechanisms of metastasis and seeking effective bio-targets for tumor metastasis to develop efficient treatment methods is inevitable.
It has been discovered that the PI3K/AKT pathway is stimulated in various cancers, strengthening tumor growth and metastasis. For example, SLC1A3 modulates the PI3K/AKT pathway, which facilitates gastric cancer progression 34 , while LncRNA HAND2-AS1 suppresses the PI3K/Akt pathway in non-small-cell lung cancer. In addition, it reduces cell proliferation and increases apoptosis 35 . While rotenone regulates the PI3K/AKT pathway to restrict colon cancer cell proliferation, motility, and EMT progress 36 , palmitic acid inactivates the pathway www.nature.com/scientificreports/ in prostate cancer to repress cell proliferation and metastasis 37 . Notably, breviscapine has been discovered to modulate the PAQR4-mediated PI3K/AKT pathway while inhibiting prostate cancer cell growth and metastasis 16 . However, the relationship between breviscapine and the PI3K/AKT pathway has not yet been investigated for CRC. This study revealed that breviscapine inactivates the PI3K/AKT pathway to inhibit CRC progression.