Ethyl acetate extract of Antenoron Filiforme inhibits the proliferation of triple negative breast cancer cells via suppressing Skp2/p21 signaling axis

Highlights

• Antenoron filiform extract inhibits triple negative breast cancer cell proliferation.

• The key target of Antenoron filiform extract is Skp2/p21 pathway.

• Quercetin is an important chemical in Antenoron filiform for the inhibition of Skp2.

Abstract

Background

Triple negative breast cancer (TNBC) has the worst prognosis of the any breast cancer subtype, and the efficient therapeutical treatment is extremely limited. Antenoron filiforme (Thunb.) Roberty & Vautier (AF) is a Traditional Chinese Medicine (TCM), which is well-known for a diverse array of pharmacological activities, including but not limited to anti-inflammatory, antioxidant and anti-tumors properties. Clinically, AF is commonly prescribed for the treatment of gynecological diseases.

Purpose

Since TNBC is one of the worst gynecological diseases, the objective of this research is to study the anti-TNBC function of the ethyl acetate extract (EAE) of AF (AF-EAE) and disclose its mechanism of action.

Materials and methods

With the aim of elucidating the underlying molecular mechanism and possible chemical basis of AF-EAE in the treatment of TNBC, a comprehensive approach combining system pharmacology and transcriptomic analysis, functional experimental validation, and computational modeling was implemented. Firstly, the potential therapeutic targets of AF-EAE treating TNBC were analyzed by systemic pharmacology and transcriptome sequencing. Subsequently, cell viability assays, cell cycle assays, and transplantation tumor assays were employed to detect the inhibitory effect of AF-EAE on TNBC. Apart from that, the western blot and RT-qPCR assays were adopted to verify its mechanism of action. Finally, the potential chemical basis of anti-TNBC function of AF-EAE was screened through molecular docking and validated by molecular dynamics.

Results

This study analyzed the differentially expressed genes after AF-EAE treatment by RNA-sequencing (RNA-seq). It was found that most of the genes were abundant in the gene set termed “cell cycle”. Besides, AF-EAE could suppress the proliferation of TNBC cells in vitro and in vivo by inhibiting the function of Skp2 protein. AF-EAE could also lead to the accumulation of p21 and a decrease of CDK6/CCND1 protein, thereby stalling the cycle of cell in the G1/S stage. Notably, clinical data survival analysis clearly demonstrated that Skp2 overexpression has been negatively correlated with survival rates in breast cancer (BC) patients. Further, as suggested by molecular docking and molecular dynamics, the quercetin and its analogues of AF-EAE might bind to Skp2 protein.

Conclusion

In summary, AF-EAE inhibits the growth of TNBC in vitro and in vivo through targeting Skp2/p21 signaling pathway. While providing a novel potential drug for treating TNBC, this study might establish a method to delve into the action mechanism of TCM.

Introduction

Nowadays, the incidence of BC increases every year and it has become the world’s most common category of cancer (Liu et al., 2022). TNBC is distinguished by the absence of the estrogen receptor (ER), human epidermal growth factor receptor 2 (HER2), and progesterone receptor (PR). As the most malignant subtype of BC, it accounts for about 10–20% of BC patients (Yin et al., 2020), and is recurrently modified in younger patients (Farheen et al., 2022). Moreover, the clinical features of TNBC include severe invasiveness, metastasis and poor prognosis. Currently, chemotherapy continues to be the main treatment strategy for TNBC (Chaudhary, 2020), but the results are not satisfactory (Denkert et al., 2017). As a consequence, developing effective treatments for TNBC treatment is critical.

In terms of the TCM, it has a time-honored history in treating cancer (So et al., 2019). TCM, alone or combined with chemotherapeutic agents, have been applied in the comprehensive treatment of cancer (Lee et al., 2014). In clinical practice, TCM is extensively used to treat BC. According to the prior research, TCM can prolong the survival time of BC patients benefited from many active ingredients, anticancer effects, and low toxicity (Fan et al., 2020). Furthermore, multiple active ingredients of TCM may have a synergistic effect by exerting multifaced effects on multiple targets simultaneously (Yang et al., 2021). Apart from that, a lot of natural products have good therapeutic effects on BC as complementary or alternative therapies. For example, ethyl gallate (Cui et al., 2015) can not only restrain the Akt-NF-κB signaling pathway, but also trigger apoptosis in BC cells. In addition, Rhein (Chang et al., 2012) anthraquinone extracted from Rheum palmatum L., is capable of inducing apoptosis as well as eliciting cell cycle arrest in BC cells. Furthermore, Saikosaponin A (Wang et al., 2020), triterpenoid glycoside isolated from Bupleurum chinense DC, exerts inhibitory effects on the proliferation and metastasis of BC both in vitro and in vivo. AF is a plant of the Polygonaceae family whose main constituents are flavonoids, phenolic acids and sterols (Lu et al., 2020; Zhao et al., 2011). Nowadays, AF is widely used for its anti-inflammatory (Huang et al., 2004), anti-coagulant, anti-tumor, analgesic and other pharmacological effects (Lu et al., 2015). However, the underlying mechanisms of anti-cancer effects of AF remain unclear and require further investigation.

It is noteworthy that the disruption of normal regulation in the cell cycle can lead to cancer. Thus, controlling the abnormal process has been regarded as an effective strategy for tumor therapy. As shown by the previous studies, the activities of cell cycle proteins, including cyclin dependent kinase (CDK) and CDK inhibitors (CDKI), could regulate the progression of cell cycle. CDKI is mainly composed of p27, p21 and p53, which negatively regulates cell cycle progression by inhibiting cell cycle proteins/CDK (Lawal et al., 2021). P21 (also known as p21RF1/CIP1 or CDKN1A), a major effector of p53, can act through p53 regulation or independently of p53. Beyond that, p21 induces a specific phase of cell cycle arrest. It is an instability protein with a considerably shorter half-life under normal conditions (Blagosklonny et al., 1996).

S-phase kinase-associated protein 2 (Skp2) serves as a pivotal regulator of cell cycle and is also recognized as a critical protein involved in the initiation of oncogenic transformation from normal cells (Li et al., 2019). Additionally, the expression of Skp2 has a negative relation to the prognosis of several cancers. The ubiquitin-proteasome pathway is the main mechanism of regulating p21 through p21 degradation, and SCF/Skp2 can induce p21 ubiquitination as well as protein hydrolysis at specific stages of the cell cycle (Blagosklonny et al., 1996). It has also been claimed that TCM can suppress the growth of tumors by hindering the cycles of cells. For example, by modulating the p53/p21/CCND1 axis, Hedyotis diffusae Herba-Andrographis Herba exhibits inhibitory effects on the proliferation of nasopharyngeal carcinoma cells through regulation of the p53/p21/CCND1 axis (Liu et al., 2022).

This research not only found out the anti-TNBC efficacy of AF-EAE both in vivo and in vitro, but also further revealed its mechanism of action by transcriptome analysis and molecular functional experiments. All in all, the finding of this research is likely to offer a therapeutic candidate for treating TNBC.