Esophageal cancer is a common malignant tumor of the digestive tract. Globally, there are about 572,000 new cases of esophageal cancer and 509,000 deaths each year [1,2]. Esophageal squamous cell carcinoma (ESCC) is one of the major subtypes of esophageal cancer, accounting for about 90 % of esophageal cancers in China. The prognosis of ESCC patients is poor, the mortality rate is high, and the 5-year overall survival rate is only 15–25 % [[3], [4], [5]]. ESCC has diverse pathogenesis, including gene mutation, RNA interference, DNA damage repair, tumor microenvironment, endoplasmic reticulum stress, immune inflammation, etc [6,7]. The treatment of ESCC mainly consists of surgery, chemotherapy, and radiotherapy, which are effective in controlling the deterioration of the disease, inhibiting the spread of cancer cells, improving the quality of life, and prolonging the survival time, but there are still problems such as many complications and adverse reactions [8]. Therefore, the search for new anti-tumor agents that are effective and less toxic has gradually become a hot research topic.

Piperlongumine (PL), also known as piplartine, belongs to alkaloids. It was originally isolated from the root of Piper longum Linn, and also found in the roots of Piper longum Roxb. and Piper tuberculatum Jacq. With the deeper research on PL, it has been found that PL has a variety of pharmacological effects. Recent studies have shown that it has specific cytotoxic effects on a variety of tumor cells, such as glioblastoma [9], Burkitt's lymphoma [10], colon cancer [11], ovarian tumor [12], prostate cancer [13], breast cancer [14], liver cancer and other malignant cancer cells [15]. It also has anti-platelet aggregation, lipid-lowering, anti-atherosclerosis, anti-inflammatory, anti-Parkinson's disease, analgesic, anti-fungal, etc., while it has no obvious toxic side-effects on normal cells, tissues, and organs [16]. PL can also reduce cancer cell viability at very low concentrations, with IC50 usually not exceeding 20 μM, and can significantly promote the apoptosis of drug-resistant cell lines [17]. This may demonstrate that PL is a highly promising natural herbal monomer capable of selectively killing tumors. Therefore, PL is a highly potential monomer of traditional Chinese medicine that can selectively kill tumors. Current studies have shown that PL exhibits cytotoxic activity against a variety of tumor cell lines and reduces tumor size in a mouse xenograft model through multiple cellular processes, including accumulation of reactive oxygen species (ROS), activation of phosphorylated AMPK, inhibition of NF-κB, and promotion of autophagy, etc [15,18]. According to the current study, PL has been reported to cause excessive accumulation of ROS in a variety of tumor cells [18,19]. Therefore, the over-accumulation of ROS may be the key to the anti-tumor effect of PL.

Recent studies have shown that PL mainly leads to the accumulation of ROS and redox abnormalities in cancer cells to promote cancer cell death. For example, Pan et al. reported that PL-induced activation of RIPK1 increased the sensitivity of cisplatin-resistant cells by stimulating mitochondrial fission to produce excessive ROS [20]. Zheng et al. confirmed that the combination of PL and sorafenib increased apoptosis in HCCLM3 and SMMC7721 cells by inducing ROS generation and mitochondrial dysfunction [15]. Zhang et al. demonstrated that PL was a novel TrxR1 inhibitor, which increased intracellular ROS levels and induced a dramatic endoplasmic reticulum stress response that contributed to cell death in hepatocellular carcinoma cells [21]. These studies suggest that PL exerts antitumor effects by inducing ROS accumulation. In addition, Li et al. found that ROS-mediated NF-κB suppression might be implicated in the mechanisms of PL-induced pyroptosis in non-small-cell lung cancer cells [22]. These studies suggest that PL can exert anti-tumour effects by inducing intracellular ROS produced by cancer cells.

Imbalance between the production and detoxification of ROS leads to oxidative stress, which significantly impacts the fate of cells. Apoptotic signaling pathway is upregulated in response to excessive ROS, facilitating normal cell death. However, aberrantly regulated biomolecules can lead to elevated ROS levels, promoting carcinogenesis in cells with impaired signaling factors. In this context, NRF2 emerges as a crucial regulator safeguarding cells against oxidative and electrophilic stress. Acting as an intracellular transcription factor, NRF2 modulates the expression of various genes involved in encoding anti-oxidative enzymes, detoxification factors, anti-apoptotic proteins, and drug transporters [23]. Experiencing oxidative stress, Keap1 undergoes critical cysteine modifications, hindering the ubiquitylation of NRF2. This modification enables Keap1 to interact with p62, facilitating its degradation through autophagy. Additionally, in quiescent cells, TXNIP is primarily located in the cytoplasm and endoplasmic reticulum (ER) and remains inactive due to its interaction with TRX. However, ROS-dependent dissociation of TRX from TXNIP leads to the translocation of TXNIP to MAMs and mitochondria, where it binds to NLRP3. This binding event triggers the activation of the NLRP3 inflammasome, resulting in augmented production of mature IL-1β [24]. The interaction between TXNIP and NLRP3 induces conformational changes in NLRP3, and knocking down TXNIP leads to increased S-nitrosylation of NLRP3 [25]. Inhibition of TXNIP through knockdown experiments results in reduced CASPASE-1 activity and decreased IL-1β release. Conversely, TRX knockdown enhances the function of inflammasomes [24,26]. These findings strongly indicate that TXNIP acts as a pivotal link connecting oxidative stress with the NLRP3 inflammasome [27]. Collectively, these studies emphasize the crucial role of the NRF2/ROS/TXNIP/NLRP3 axis in mediating oxidative stress. Moreover, ROS has been shown to stimulate pyroptosis. Combined with previous literature, we speculate that excessive ROS production may be responsible for PL-induced pyroptosis. However, no studies have been reported on the antitumor effects of PL on ESCC. Therefore, the present study aimed to explore the anticancer effects of PL on ESCC cells in vitro and in vivo and further investigate the underlying mechanisms.