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Curcumin against gastrointestinal cancer: A review of the pharmacological mechanisms underlying its antitumor activity

Gastrointestinal cancer (GIC) poses a serious threat to human health globally. Curcumin (CUR), a hydrophobic polyphenol extracted from the rhizome of Curcuma longa, has shown reliable anticancer function and low toxicity, thereby offering broad research prospects. Numerous studies have demonstrated the pharmacological mechanisms underlying the effectiveness of CUR against GIC, including the induction of apoptosis and autophagy, arrest of the cell cycle, inhibition of the epithelial–mesenchymal transition (EMT) processes, inhibition of cell invasion and migration, regulation of multiple signaling pathways, sensitization to chemotherapy and reversal of resistance to such treatments, and regulation of the tumor survival environment. It has been confirmed that CUR exerts its antitumor effects on GIC through these mechanisms in vitro and in vivo. Moreover, treatment with CUR is safe and tolerable. Newly discovered types of regulated cell death (RCD), such as pyroptosis, necroptosis, and ferroptosis, may provide a new direction for research on the efficacy of CUR against GIC. In this review, we discuss the recently found pharmacological mechanisms underlying the effects of CUR against GIC (gastric and colorectal cancers). The objective is to provide a reference for further research on treatments against GIC.

Introduction
Gastrointestinal cancer (GIC) is characterized by high incidence and mortality rates, posing a serious threat to human health globally. According to the Global Cancer Statistics 2020 estimates (Sung et al., 2021), gastric cancer (GC) ranked fifth and fourth among cancers in terms of incidence (>1 million new cases) and mortality (769,000 deaths), OPEN ACCESS EDITED BY respectively. Similarly, colorectal cancer (CRC) accounts for >1.9 million new cases and 935,000 deaths, ranking third and second among cancers, respectively.
Surgical treatment, radiotherapy, and chemotherapy have traditionally been the main strategies for the treatment of GIC. However, owing to the low rate of early detection and high rate of postoperative recurrence associated with GIC, the effectiveness of radiotherapy and chemotherapy may be compromised by the occurrence of severe undesirable side effects. In the search for effective treatments with fewer side effects, an increasing number of research studies are focused on traditional herbal medicines and their monomer compounds.
Curcumin (CUR) was first discovered by Vogel and Pelletier (Prasad et al., 2014), and is the most important component of the rhizomes of turmeric (Curcuma longa) (Waly et al., 2018). CUR is a hydrophobic polyphenol that has been approved by the US Food and Drug Administration based on its bio-safety (Mashayekhi-Sardoo et al., 2021). Moreover, it has demonstrated a wide range of pharmacological activities, such as antibacterial (Ibarra-Martinez et al., 2022), anti-inflammatory , antioxidant (Xu et al., 2021), and antitumor . CUR has been widely reported to inhibit the proliferation of tumor cells in a concentration-and timedependent manner in vitro (Li et al., 2017a;Fan et al., 2020;Mao et al., 2021). According to relevant clinical trials on safety and toxicity, the acceptable dose of CUR for maximum efficacy is 4-8 g per day. It has been reported that humans can tolerate treatment with CUR at a dose up to 12 g per day (Barati et al., 2019).
In this review article, we discuss the pharmacological mechanisms underlying the effects of CUR against GIC based on recent evidence derived from in vitro and in vivo experiments, as well as clinical trials ( Figure 1). The objective of this review is to provide a reference for further research on the treatment of GIC using CUR.
2 Curcumin in gastrointestinal cancer (gastric cancer and colorectal cancer)

Inhibition of gastrointestinal cancer cell proliferation and blockage of cell cycle
It is widely recognized that CUR inhibits the proliferation and cell cycle of GIC cells. Cyclin D1 (CCND1) and cyclindependent kinase 4 (CDK4), a serine/threonine kinase which binds to CCND, can regulate cell transition from G1 to S phase (Yin et al., 2018). Sun et al. (Sun et al., 2019) found that CUR significantly inhibited cell cycle progression of SGC7901 cells in the G0/G1-S phase, increased the cell number in the G0/ G1 phase, and downregulated the expression of B-cell lymphoma 2 (Bcl-2), CDK4, and CCND1 proteins in both cells and tissues.
Herrero et al. (De La Parte et al., 2021) found that CUR inhibited the proliferation of CRC cells CC531 in vitro and reduced the tumor volume in liver implants in vivo. Mao et al. reported that CUR inhibited tumorsphere formation and reduced cell viability in a concentration-dependent manner in LGR5 (+) colorectal cancer stem cells (CSCs). Inhibition of the proliferation of HCT-116 and LoVo CRC cell lines by CUR has also been reported (Mao et al., 2021). Blakemore et al. (Blakemore et al., 2013) elucidated the stage of arrest induced by CUR in eight CRC cell lines (i.e., Caco-2, DLD-1, HCA-7, HCT116p53+/+,HCT116p53 −/− , HCT116p21 −/− , HT-29, and SW480). The results showed that the majority of these eight cell lines were arrested at the G2/M transition, with some cells arrested in mitosis. Notably, HCT116 and Caco-2 cells presented the highest levels of mitotic arrest.

Induction of cell apoptosis
Apoptosis is an indispensable mechanism for maintaining normal and healthy bodily functions. Any change in apoptotic patterns can enhance the survival and progression of cancer cells (Li et al., 2017b;Liang et al., 2018). CUR can promote the apoptosis of tumor cells by regulating multiple signaling pathways, upregulating proapoptotic genes, or downregulating antiapoptotic genes, thus inhibiting cancer progression (Liang et al., 2018).

Activation of the p53 signaling pathway
The tumor suppressor gene p53 plays an important role in the pathology of human cancer. The protein p53 inhibits the growth of cancer cells by regulating cell cycle arrest, cell apoptosis, and DNA repair (Hafner et al., 2019). Upregulation of p53 expression inhibits the proliferation and induces apoptosis of cancer cells (Su et al., 2017).
It has been shown that treatment with CUR for 24 h inhibited the proliferation and apoptosis of GC cells SGC-7901 and BGC-823 in a dose-dependent manner. Further studies found that this effect was related to the activation of the p53 signaling pathway through the upregulation of p53 and p21 (Fu et al., 2018).
By taking advantage of the waiting period before surgery, He et al. (2011) treated a group of CRC patients with CUR. The results showed that weight loss was improved in patients treated with CUR. In addition, by analyzing in human CRC tissue specimens obtained at the time of diagnosis (biopsy via colonoscopy) and surgery (surgical removal of CRC tissue), they demonstrated that CUR reduced the serum levels of TNF-α, induced apoptosis of CRC cells, upregulated p53 expression, and regulated the expression of apoptosisrelated Bax and Bcl-2.

Inhibition of the phosphoinositide-3kinase/Akt (PI3K/Akt) signaling pathway
The PI3K/Akt/mechanistic target of rapamycin kinase (PI3K/Akt/mTOR) signaling pathway is a well-studied antiapoptotic and pro-proliferative signaling pathway (Braglia et al., 2020). It has been revealed that this pathway is activated in approximately one-third of human cancers (Khorasani et al., 2021). Abnormal activation and overexpression of key signal molecules in the PI3K/Akt signaling pathway may promote the occurrence and development of GIC.
By treating SGC-7901, BGC-823, and MKN28 cell lines with increasing amounts of CUR for 24, 48, and 72 h, Li et al. (2017a) found that CUR could inhibit the proliferation and induce apoptosis of GC cells in a time-and dose-dependent manner. The results of western blotting assays showed that treatment with CUR downregulated the expression of phospho-Akt together with that of downstream phospho-mTOR and phospho-p70s6K in a dose-dependent manner. These findings indicated that the activation of the PI3K/Akt/mTOR signaling pathway was suppressed in GC cells following treatment with CUR. Liu et al. (2018) reported that CUR could inhibit the PI3K/Akt signaling pathway and activate the activity of p21 protein, thus promoting apoptosis in GC cell line AGS.
It has been reported that CUR downregulated PI3K and Akt expression while promoting apoptosis in the early stages of CRC induced by 1,2-dimethylhydrazine dihydrochloride in rats (Rana et al., 2015). Also, the results of western blotting assays showed that CUR upregulated the expression of pro-apoptotic Bcl-2 family members (Bcl-2 associated agonist of cell death [Bad] and Bax) and cysteine protease family apoptosis executioner (CASP3/9), whereas it downregulated that of antiapoptotic Bcl-2 protein.

Inhibition of the Wnt/β-catenin signaling pathway
β-catenin can be activated by Wnt signaling and is involved in different types of signal transduction. Studies have confirmed that it is essential for tumor growth (Zheng et al., 2017). The regulation of the Wnt/β-catenin signaling pathway is highly relevant to the metabolism of cancer cells (Cantor & Sabatini, 2012), and plays an important role in the occurrence, progression, and prognosis of various types of cancer in humans. Activation of the Wnt/β-catenin signaling pathway results in the development of resistance to chemotherapy in several types of cancer (Sun et al., 2012;Nagaraj et al., 2015). Therefore, it is hypothesized that it could effectively inhibit tumor growth by targeting the Wnt/β-catenin signaling pathway (Zheng et al., 2017). Zheng et al. (2017) found that CUR significantly attenuated cell viability and induced tumor cell apoptosis in vitro in GC cell Frontiers in Pharmacology frontiersin.org 03 lines SNU-1, SNU-5, and AGS. CUR significantly suppressed the levels of Wnt3a, LDL receptor related protein 6 (LRP6), phospho-LRP6, β-catenin, phospho-β-catenin, c-Myc, and survivin, demonstrating that Wnt/β-catenin signaling was downregulated. In an in vivo experiment, AGS cells were used to establish a xenograft tumor model in male BALB/c nude mice. The CUR treatment group received CUR (1 mg/kg body weight) through gavage once daily, and the results showed that xenograft growth was inhibited by treatment with CUR.
The Wnt/β-catenin signaling pathway has been considered one of the most frequently dysregulated pathways in CRC (McCubrey et al., 2016). Its abnormal activation is related to cell proliferation, invasiveness, and resistance to therapy, indicating its potential value as a target in the treatment of CRC (Bahrami et al., 2017). Li et al. (2018) found that high levels of paternally expressed gene 10 (PEG10) increased the expression of Wnt1 and β-catenin in colon tissues, and this effect may be regulated by miR-491. By upregulating miR-491 and inhibiting the Wnt/β-catenin signaling pathway, CUR inhibited the expression of PEG10, thereby inducing apoptosis and impairing the proliferation of HCT-116 cells.
Axin2, a downstream gene of the Wnt/β-catenin signaling pathway, has been associated with cell proliferation, mutation, migration, and apoptosis (Gunes et al., 2010;Aristizabal-Pachon et al., 2015). Hao et al. (Hao et al., 2021) found that the Wnt/βcatenin pathway was activated in human CRC samples, accompanied by an increased mRNA expression of Axin2. Furthermore, they demonstrated that CUR could reduce Axin2 in the Wnt/β-catenin pathway, thus inhibiting CRC in vivo.

Induction of reactive oxygen species production
Reactive oxygen species (ROS), a general term describing oxygen or oxygen-containing highly reactive molecules, acts as a doubleedged sword for cancer cells; these molecules act differently at different stages of cancer (Prasad et al., 2017). ROS can promote the proliferation and growth of cancer cells by activating a variety of cell signaling pathways, while the excessive accumulation of ROS can lead to cell damage and apoptosis . Tong et al. (Tong et al., 2020) found that high concentrations of CUR (≥20 μmol/L) increased the levels of ROS in human GC cells, leading to mitochondrial damage, DNA damage, and apoptosis. These findings indicated that the anticancer effect of CUR in GC may be closely related to its pro-oxidative effect at high concentrations and elevation in the levels of ROS in cancer cells.
In addition, the investigators demonstrated that CUR induced DNA demethylation in human GC cells through the damaged DNA repair-p53-p21/growth arrest and DNA damage inducible alpha-cyclin (GADD45A-cyclin)/CDK-retinoblastoma/E2F-DNA methyltransferase1 (E2F-DNMT1) axis. Liang et al. (2014) found that CUR induced apoptosis combined with the production of ROS in BGC-823 cells. The apoptosis induced by CUR was reduced by adding an antioxidant (N-acetyl cysteine [NAC] or trion) to inhibit ROS production. Additional experiments showed that the apoptotic signal regulating the kinase 1-mitogen-activated protein kinase 4-JUN N-terminal kinase (ASK1-MKK4-JNK) signaling pathway was activated by oxidative stress induced by CUR, further leading to apoptosis. Sritharan & Sivalingam (2021) found that treatment with CUR induced apoptosis, ROS generation, and a decrease in MMP in HT-29 and HCT-116 cell lines. Pre-treatment with NAC effectively reduced the levels of ROS and prevented MMP loss in HT-29 cells; however, it was less effective in HCT-116 cells. Pre-treatment of HCT-116 cells with NAC for 24 h upregulated the protein expression of total p53. This evidence demonstrated that CUR induced ROS-mediated apoptosis in CRC cell lines, and this effect may be mediated by p53. Liu et al. (2014) treated SGC7901 cells with coadministration of CUR at serial concentrations and diazoxide-an adenosine triphosphate-sensitive (ATP-sensitive) potassium (KATP) channel opener. They found that CUR induced the opening of KATP channels, leading to loss of the mitochondrial membrane potential (MMP) in a dose-dependent manner; moreover, it induced apoptosis of tumor cells.

Activation of the mitochondrial pathway
Inhibition of glycolysis has been a therapeutic target in cancer (Hamanaka & Chandel, 2012). The regulation of key glycolytic enzymes, such as hexokinase 2 (HK2), is an effective approach to the inhibition of glycolysis (Patra et al., 2013). Wang K. et al (2015) found that the uptake of glucose, as well as the production of ATP and lactic acid, were inhibited by CUR in a dose-dependent manner in human CRC cells HCT116 and HT29. Further study showed that CUR induced the dissociation of HK2 from the mitochondria and triggered mitochondria-mediated apoptosis. Collectively, the evidence demonstrated that CUR inhibited glycolysis and promoted mitochondria-mediated apoptosis by regulating HK2 in CRC cells.
Similarly, it has been reported that CUR induced apoptosis through the mitochondrial cell death pathway in LoVo cells (Guo et al., 2013). Guo et al. found that CUR induced apoptosis in LoVo cells, accompanied by the release of lactate dehydrogenase, collapse of MMP, arrest of the cell cycle, and activation of CASP3/9. Moreover, the results of western blotting analysis showed that CUR upregulated the expression of cytochrome c, Bax, and p53, whereas it downregulated that of Bcl-2 and survivin in LoVo cells.

Induction of autophagy
Autophagy is a catabolic degradation process in which cellular proteins or organelles are degraded in the lysosome Frontiers in Pharmacology frontiersin.org 04 and recycled. This process plays an important and complicated role in the occurrence and development of cancer, as well as the development of resistance to therapy (Li et al., 2017a). Studies utilizing transmission electron microscopy revealed the presence of autophagosomes in GC cells (SGC-7901 and BGC-823) treated with CUR (Fu et al., 2018). Li et al. (2017a) found that CUR induced the formation of acidic vesicular organelles, conversion of LC3-I to LC3-II, and upregulation of the expression of autophagy-related proteins, such as beclin 1 (BECN1), autophagy related 7 (ATG7), and ATG5-12 conjugate in BGC-823, SGC-7901, and MKN-28 cells. These results demonstrated that CUR induced autophagy in GC cells. Interestingly, CUR and autophagy inhibitor 3methyladenine co-treatment attenuated the viability of GC cells and increased apoptosis. In conclusion, they suggested that CUR induced a protective autophagy, which could be antagonistic apoptotic cell death in GC cells. Mao et al. (2021) found that CUR induced autophagy in colorectal CSCs in a concentration-dependent manner. The results obtained from transmission electron micrographs confirmed this finding. In addition, co-treatment with hydroxychloroquine (an autophagy inhibitor) significantly decreased the cell proliferation induced by CUR.

Inhibition of epithelial-mesenchymal transition processes
By converting the epithelial characteristics of cells into mesenchymal attributes, EMT stimulates the progression of cancer. This is achieved by enhancing the invasive, migratory, and metastatic abilities of cancer cells. In recent years, studies have focused on elucidating the mechanism involved in the inhibition of EMT by treatment with CUR in GIC cells (Suarez-Carmona et al., 2017).

Inhibition of the Wnt/β-catenin pathway
The Wnt/β-catenin signaling pathway, an important pathway in EMT, plays an important role in embryogenesis and human diseases, including various types of cancer. Zhang et al. (2020) reported that CUR inhibited EMT processes, cell migration, invasion, and cytoskeletal remodeling, and induced apoptosis in SGC-7901 cells. The results of reverse transcription-quantitative polymerase chain reaction, western blotting, and co-immunoprecipitation analyses showed downregulation of the mRNA and protein expression of sonic hedgehog signaling molecule (SHH), GLI family zinc finger 1 (GLI1), and forkhead box M1 (FOXM1) in the SHH signaling pathway, and β-catenin in the Wnt signaling pathway. These findings indicated that CUR inhibits the SHH and Wnt signaling pathways. This inhibition of the aforementioned signaling pathway, as well as the addition of CUR, also suppressed the EMT process. In addition, the evidence demonstrated an interaction between GLI1 and β-catenin, which could be inhibited by CUR. Zhang et al. (2016) found that CUR inhibited EMT in CRC cells via the naked cuticle homolog 2-Wnt-C-X-C motif chemokine receptor 4 (NKD2-Wnt-CXCR4) signaling pathway. The results showed that CUR significantly inhibited the proliferation of CRC cells and upregulated the expression of NKD2 in SW620 CRC cells. These changes resulted in the regulation of key markers (downregulation of β-catenin and transcription factor 4 [TCF4] expression, upregulation of axin) of Wnt signaling. In addition, the progression of EMT was inhibited through the overexpression of E-cadherin and downregulation of vimentin. CUR also inhibited tumor metastasis by significantly reducing the expression of CXCR4. Chen et al. (2020) reported that CUR downregulated the expression of transcription factors to promote EMT in CRC cells by reducing methylation in the promoter of caudal type homeobox 2 (CDX2) and inhibiting the CDX2/Wnt3a/βcatenin signaling pathway. In human SW480 CRC cells, CUR inhibited EMT, accompanied by the downregulation of DNMT1 and DNMT3a expression, and reduction of the methylation levels in the CDX2 promoter in a concentrationdependent manner. Treatment with CUR reduced the expression levels of N-cadherin, vimentin, Wnt3a, Snail1, and Twist, as well as the nuclear translocation levels of β-catenin in a concentration-dependent manner. In contrast, the expression levels of E-cadherin were increased.

Regulation of miR-200c/EPM5
The tumor suppressor miR-200c is upregulated by CUR in CRC cells (SW620 and HT29) . As a consequence of EMT repression, cell migration and invasion were also inhibited by CUR. Treatment with CUR also downregulated the expression of EPM5 (also termed PRICKLE2), a direct target of miR-200c. Further investigation indicated that EPM5 acted as a downstream of the CUR/miR-200c cascade, and miR-200c mediated the CUR-repressed EMT by direct repression of EPM5.

Inhibition of invasion and migration
Metastasis of a malignant tumor is often the main reason for failure of therapy. Therefore, an important part of the research on anticancer drugs is focused on the inhibition of tumor metastasis.
Numerous studies Sun et al., 2019;Tong et al., 2020;Zhang et al., 2020) have demonstrated that CUR could inhibit the invasion and migration of GC cells. Liu et al. reported that CUR inhibited the expression of miRNA-21 and decreased that of matrix metalloproteinase 2 (MMP2) and MMP9, thus inhibiting the invasion and migration of AGS cells . Zhang et al.  found that CUR affected the migration, invasion, and cytoskeletal Frontiers in Pharmacology frontiersin.org remodeling of SGC-7901 cells by regulating the GLI1-β-catenin pathway.
In vitro and in vivo studies have shown that CUR inhibited the invasion, metastasis, and angiogenesis of CRC cells Calibasi-Kocal et al., 2019;De La Parte et al., 2021). According to Chen et al. (Chen et al., 2013), the antimetastatic effect of CUR on CRC cells is related to the downregulation of Sp-1 (transcription factor), FAK (cell adhesion component), and CD24, as well as the upregulation of E-cadherin expression.
Herrero et al. (De La Parte et al., 2021) reported the antitumor effect of CUR against liver implants from CRC both in vitro and in vivo. Importantly, the investigators quantified the total and individual liver lobe tumor volume in untreated and CUR-treated WAG/RijHsd tumor-bearing rats. They found that CUR reduced the tumor volume in liver implants. Similar findings ) in a mouse model have also been reported.
2.6 Sensitization to chemotherapy and reversal of resistance to chemotherapy 2.6.1 Gastric cancer Chemotherapy, one of the most important treatment options for GC, greatly improves the prognosis and survival of patients with GC. However, the occurrence of chemotherapy-resistant cancer cells has resulted in treatment failure or disease recurrence. Furthermore, the severe adverse effects caused by chemotherapy drugs negatively impact patient quality of life. CUR is a natural plant extract with a good safety profile. Hence, its role in sensitizing cancer cells to chemotherapy and reversing resistance to treatment has become an important topic of research in the GC setting. Drugs commonly used in chemotherapy against GC include 5-fluorouracil (5-FU), cisplatin (DDP), doxorubicin (DOX), etc.
In a study, AGS cells were treated with CUR, DOX, and their combination (DOX-CUR). The results showed that all these treatments significantly inhibited the viability, tumor spheroid formation, migration, and invasion in AGS cells. Notably, the DOX-CUR combination exhibited a more significant anticancer activity than monotherapy with CUR or DOX (Amoodizaj et al., 2020). Yu et al. (2011) found that chemotherapeutics (etoposide and DOX) induced apoptosis and activated NF-κB in SGC-7901 cells. The combination of CUR and chemotherapeutics induced apoptosis, while the activation of NF-κB was attenuated and the expression of the downstream antiapoptotic genes Bcl-2 and Bcl-xL was downregulated.
By establishing a 5-FU-resistant GC cell line (SGCR/5-FU), Kang et al. (2016) found that cytosolic IκBα degradation and NF-κB nuclear translocation were increased in these cells. Treatment with CUR reversed 5-FU resistance and inhibited the proliferation of GC cells by inhibiting the NF-κB signaling pathway. Moreover, downregulation of tumor necrosis factorα (TNF-α) mRNA was induced by CUR in GC cells.
A regimen combining 5-FU with DDP (FP) is typically used in chemotherapy against GC. He et al. (2017) found that CUR enhanced the effects of treatment with FP on the viability, colony formation, and migratory abilities of MGC-803 cells. This effect may be associated with the apoptosis induced by CUR via the CASP3/8, Bcl-2, and Bax signaling pathways.
The chemotherapeutic regimen combining 5-FU, folinic acid, plus OXA (FOLFOX) plays an important role in the treatment of GC. Nevertheless, it is associated with extensive side effects and the development of resistance in some patients (Kim et al., 2011). Zhou et al. (2016) explored the synergistic antitumor effect of CUR and FOLFOX against GC in vitro and in vivo. Using cell proliferation assays and assessment of apoptosis, they demonstrated the synergistic antitumor effect of CUR in combination with 5-FU and OXA (5-FU/OXA). This effect may be associated with the downregulation of Bcl-2 expression and upregulation of Bax and CASP3/8/9 expression caused by CUR. In addition, the in vivo data showed that the combination of CUR and 5-FU/OXA significantly inhibited the growth of BGC-823 xenograft tumors.

Colorectal cancer
Chemotherapy is an important therapeutic modality in the treatment of CRC. However, toxicity and the development of resistance limit its efficacy. CUR could synergize with DDP to inhibit CRC cells and effectively suppress the occurrence of resistance to DDP in those cells (Zheng Z.-h. et al, 2021;Fan et al., 2022). Fan et al. (2022) reported that this effect of CUR was achieved by targeting the miRNA-137-glutaminase axis. They found that DDP-resistant CRC cells (HT-29) displayed an increased glutamine metabolism, and this elevation could be attenuated by CUR. Further study showed that miR-137, which directly targets glutaminase, was induced by treatment with CUR, leading to sensitization of CRC cells to DDP. According to a study conducted by Zheng et al., the reversal effect of CUR on resistance to DDP in CRC cells was based on the downregulation of lncRNA KCNQ1 opposite strand/antisense transcript 1 (KCNQ1OT1) expression. KCNQ1OT1 is upregulated in HCT8/DDP cells and regulates their proliferation and apoptosis by blocking the suppressive effect of miR-497 on Bcl-2 expression. In vitro and vivo experiments showed that CUR reversed the development of resistance to DDP in CRC by downregulating KCNQ1OT1 expression, thereby regulating the miR-497/Bcl-2 axis.
It was reported that >15% of patients are resistant to treatment with 5-FU-based chemotherapeutic regimens (Shakibaei et al., 2014). Thus, the resistance to 5-FU is also a research focus in the CRC setting. Studies (Shakibaei et al., 2014;Zhang et al., 2018;Lu et al., 2020; confirmed that CUR could enhance the sensitivity of CRC cells to 5-FU and reverse the occurrence of resistance. Nicotinamide N-methyltransferase (NNMT) is highly expressed in a wide variety of cancers. It has been reported that NNMT could Frontiers in Pharmacology frontiersin.org enhance resistance to 5-FU in human CRC cells (Xie et al., 2016;Li G. et al, 2021). Li et al. (Li G. et al, 2021) found that the effect of CUR on reversing NNMT-induced resistance to 5-FU was related to ROS production and cell cycle arrest. They demonstrated that CUR inhibited the expression of NNMT, though the precise mechanism involved in this process is unclear. Zheng X. et al (2021) showed synergistic inhibitory effects of CUR and 5-FU both in vivo and in vitro in CRC SW620 cells. These effects may be attributed to inhibition of STAT1 activation to reduce L1 expression and induce a higher level of cell apoptosis. By establishing the 5-FU resistant HCT-116 cell line, Lu et al. (2020) found that CUR reversed resistance to 5-FU in CRC cells through inhibition of the EMT process. This effect may be achieved by regulation of the TET1-NKD-Wnt pathway. Zhang et al. (2018) reported the important role of nuclear factor erythroid 2-related factor (Nrf2) in the effect of CUR on the reversal of resistance to 5-FU in CRC. CUR suppressed the mRNA and protein expression of Nrf2, which further reduced the expression levels of Bcl-2. These effects promoted apoptosis in HCT-8/5-FU cells and reversed MDR. OXA is a platinum-based chemotherapeutic agent that plays an important role in the treatment of CRC (Yin et al., 2019); nevertheless, the development of resistance limits its efficacy. Han et al. (2020) established the OXA-resistant CRC cell line HCT116/ OXA, and found that CUR could reverse resistance to its OXA. Furthermore, they suggested that the mechanism underlying this effect may be the inhibition of excision repair cross-complementation 1 (ERCC1) through upregulation of miRNA-409-3p. These alterations result in inhibition of the expression of drug resistance-related proteins [Bcl-2, glutathione S-transferase-π (GST-π), mitochondrial ribosomal protein (MRP), P-gp, and survivin]. Yin et al. (2019) also established an OXA-resistant cell line (HCT116/OXA) to investigate the effect of CUR on resistance to OXA in CRC. According to the results, CUR could reverse resistance to OXA in CRC through inhibition of EMT by suppressing the transforming growth factor-β/Smads (TGF-β/Smads) pathway both in vitro and in vivo. TGF-β, regulated by Smad and non-Smad signaling pathways, is an important inducer of EMT (Katsuno et al., 2013). EMT has been gradually recognized as an important mechanism in the development of drug resistance in tumors (Yin et al., 2019). Ruiz et al. (Ruiz de Porras et al., 2016) found that NF-κB was overactivated in OXA-resistant CRC cells, and this overactivation could be attenuated by treatment with CUR. Meanwhile, they found that treatment with CUR plus OXA could revert resistance to OXA, potentially through inhibition of the NF-κB signaling cascade. In addition, the investigators reported that C-X-C motif chemokine ligand 1 (CXCL1) was regulated by NF-κB; its expression was upregulated in resistant cells, but downregulated after treatment with CUR plus OXA. Thus, they speculated that CXCL1 could be used as a predictive marker in patients with CRC. The majority of studies on CRC cells are conducted in vitro. Guo et al. (Guo L.-d. et al, 2015) evaluated the therapeutic effect of CUR and OXA in vivo by establishing xenografts through subcutaneous implantation of LoVo CRC cells in nude mice. The results showed that the combinatorial administration of CUR and OXA inhibited the growth of CRC in nude mice, caused cell cycle arrest, and induced apoptosis. In addition, some important signaling molecules were tested; the results showed that the expression of Bax, CASP3, and PARP was significantly increased, whereas that of Bcl-2, survivin, HSP70, pro-CASP3, and pro-PARP was markedly suppressed in tumor cells. Su et al. (2018) also reported that CUR could reduce resistance to irinotecan (CPT-11). LoVo/CPT-11, an established CPT-11-resistant CRC cell line, exhibited markedly higher expression levels (mRNA and protein) of colon CSC markers (CD44, CD133, epithelial cell adhesion molecule [EpCAM], and CD24) compared with parental cells. Furthermore, treatment with CUR attenuated resistance to CPT-11, accompanied by reduction in the expression levels of CSC identification markers. In addition, they found that CUR induced cell apoptosis in sphere-forming cells, and the combination of CUR and CPT-11 could increase the apoptosis-inducing effect.
FOLFOX is the most commonly used combination chemotherapeutic regimen for CRC (Al Sabbagh et al., 2021). In recent years, studies (including clinical trials) on CUR and FOLFOX have been conducted. James et al. (James et al., 2015) established ex vivo CSC models from patient-derived colorectal liver metastases. They found that the combination of CUR and 5-FU/OXA could significantly reduce the number of spheroids, while the levels of CSC markers (ALDH and CD133) were also decreased. A phase I clinical trial (James et al., 2015) performed in the United Kingdom in patients with liver metastases from CRC showed that addition of daily oral CUR (0.5-2 g) to FOLFOX chemotherapy is safe and tolerable. A phase IIa (Howells et al., 2019), two-armed, randomized, and controlled clinical trial further demonstrated the safety and tolerability of this combination in patients with metastatic CRC who were randomly assigned to receive either FOLFOX or CUFOX (FOLFOX +2 g oral CUR per day). A significant difference in overall survival was observed between patients receiving FOLFOX and CUFOX. The concentration of plasma CXCL1 [growth-regulated oncogene-α (GRO-α)], which is associated with metastatic spread in CRC, was 1.7-fold higher in the FOLFOX group versus the CUFOX group; however, the difference was not statistically significant. Hence, it is suggested that the combination treatment has the potential to provide benefit to patients, though the currently available evidence is insufficient. Both phase I and II trials involved a small number of patients (12 and 28, respectively) and lacked a placebo control group. Thus, the clinical efficacy of CUR as an adjunct of FOLFOX chemotherapy is uncertain, and phase III trials are warranted.

Curcumin in gastric cancer
The unique anti-GC mechanism of CUR, which is described in detail below, combined with the above common anti-GIC mechanism of CUR is illustrated in Figure 2 and summarized in Table 1.
Frontiers in Pharmacology frontiersin.org

Activation of caspase 3 signaling pathway and regulation of Bcl-2associated X, Bcl-2
Activation of the cysteine protease (caspase) family plays a critical role in apoptosis and is considered a direct effector of apoptosis. Caspase 3 (CASP3) is the main effector molecule of apoptosis; it can lyse DNA repair-related molecules, apoptosis inhibitory proteins, extracellular matrix proteins, and skeleton proteins. The activation of CASP3 signals an reliable marker for apoptosis (Crowley & Waterhouse, 2016). Members of the Bcl-2 family play a central role in the regulation of apoptosis and influence the cell cycle, particularly the transition from quiescence to proliferation (Xu et al., 2011). He et al.  demonstrated that CUR inhibited the proliferation and promoted apoptosis of MGC803 cells via the activation of CASP3/8, downregulation of Bcl-2, and upregulation of Bcl-2-associated X (Bax).

Downregulation of miRNA-21
It has been found that miRNA-21 is highly expressed in GC tissues . CUR induced the activity of phosphatase and tensin homolog (PTEN) and inhibited that of the PI3K/Akt signaling pathway by inhibiting miRNA-21 expression in AGS cells. These effects resulted in the inhibition of biological activity and induced

Model(s) Cell line(s) Dose(s) Mechanism(s) References
In apoptosis of AGS cells. Similar findings were also obtained in MGC803 cells . Qiang et al. (Qiang et al., 2019) found that CUR induced apoptosis, inhibited the expression of phospho-Akt protein, upregulated PTEN expression, and downregulated the levels of miRNA-21 in MGC803 cells. These results suggested that CUR negatively modulated the miR-21/PTEN/ Akt pathway in MGC803 cells.

Downregulation of long non-coding RNA H19
It is gradually accepted that long non-coding RNA (lncRNAs) play multiple roles in the progression of cancer. Previous studies (Song et al., 2013;Wang J. et al, 2015) have reported that H19 is abnormally upregulated in GC. This upregulation directly inhibits p53 activation, thereby promoting GC progression (Yang et al., 2012). Liu et al. (Liu et al., 2016) found that CUR inhibited the expression of H19 and upregulated that of p53 in a concentrationdependent manner in SGC7901 cells. Further investigation showed that CUR downregulated the expression of the c-Myc oncogene. In addition, the downregulation of H19 expression induced by CUR could be reversed by exogenous c-Myc protein. This observation suggested that CUR inhibited the proliferation and induced apoptosis of GC cells by downregulating the c-Myc/H19 pathway.

Upregulation of miR-34a
It has been revealed that miR-34a plays a critical role in GC (Wong et al., 2011). Sun et al. (Sun et al., 2019) found that CUR could upregulate miR-34a expression, inhibit proliferation, and induce apoptosis in SGC7901 cells. Administration of CUR or miR-34a in GC cells or tissues decreased the expression of Bcl-2, CDK4, and CCND1. It is suggested that CUR may downregulate Bcl-2, CDK4, and CCND1 expression by upregulating miR-34a, thus inducing apoptosis in GC. Cao et al. (2013) indicated that CUR could function through endoplasmic reticulum (ER) stress and mitochondria functional pathways in GC cells AGS, based on the upregulation of CCAAT/ enhancer binding protein homologous protein (CHOP), phosphorylation of JNK and downregulation of SERCA2ATPase, release of cytochrome c, reduction of Bcl-2, and loss of MMP.

Suppression of gastric acid secretion
The development of GC is often accompanied by excessive gastric acid secretion (Deng et al., 2012). It is widely thought that the acidic environment in the stomach contributes to the development of GC (Sheng et al., 2018). Therefore, hyperacidity may be related to the progression of GC. Gastrin, an inducer of gastric acid secretion, is a valuable marker for GC screening (Waldum et al., 2017). Zhou et al. (2017) evaluated the mechanism through which CUR inhibits GC by affecting gastric acid secretion both in vitro and in vivo. The results showed that

Model(s) Cell line(s) Dose(s) Mechanism(s) References
In Frontiers in Pharmacology frontiersin.org CUR significantly inhibited GC progression through an increase in gastric pH and suppression of gastrin-mediated acid secretion.

Inhibition of angiogenesis and lymphangiogenesis
Vascular endothelial growth factor (VEGF) can promote angiogenesis and growth of cancer cells, and is considered the most important and specific angiogenic factor. Signal transduction and transcription activator 3 (STAT3) is highly expressed in GC, and is closely related to cancer cell stage, invasion depth, lymph node metastasis, and cancer grade. Overexpression of STAT3 and VEGF in GC cells induced an increase in microvascular density and disease progression. Using BALB/C mice grafted with a mouse gastric adenocarcinoma cell line MFC as an experimental model, Wang et al. (2017) investigated the antitumor activity of CUR. The results showed that CUR inhibited the proliferation of GC by downregulating the differentiated embryonic chondrocyteexpressed gene 1-hypoxia inducible factor 1 subunit alpha-STAT3-VEGF (DEC1-HIF1A-STAT3-VEGF) signal transduction pathway. Huang et al. (2017) reported that tumor-derived mesenchymal stem cells (MSC) are important for tumor angiogenesis, and investigated the mechanism through which CUR mediates angiogenesis to regulate GC cell-derived MSC (GC-MSC). The results showed that CUR reduced GC-MSCderived angiogenesis by inhibiting the NF-κB/VEGF signaling pathway, which plays a critical role in tube formation, migration, and colony formation induced by GC-MSC.
High mobility group box 1 (HMGB1) and vascular endothelial growth factor D (VEGFD) are associated with lymphangiogenesis and tumor metastasis in GC. Da et al. (2019) found that CUR may inhibit lymphangiogenesis in GC cells (AGS and SGC-7901) through the inhibition of HMGB1/ VEGFD signaling. In addition, they investigated the effect of CUR on lymphatic vessel density in a nude mouse model using SGC7901 cells. The results indicated that CUR inhibited lymph node metastasis of GC, as evidenced by the reduced tumor volume, decreased protein expression of prospero homeobox 1 (PROX1), podoplanin (PDPN), and vascular endothelial growth factor receptor 3 (VEGFR3), and downregulated mRNA expression of lymphatic vessel endothelial receptor 1 (LYVE1), PROX1, PDPN, and VEGFR3 (Da et al., 2015).

Curcumin in colorectal cancer
The anti-CRC mechanism of CUR, which is described in detail below, combined with the above common anti-GIC mechanism of CUR is illustrated in Figure 3 and summarized in Table 2.

Downregulation of CD44 expression
CD44, a family of transmembrane glycoproteins, is an important cancer stem cell marker in CRC. It is involved in multiple cellular biological functions, such as proliferation, migration, and survival (Thapa & Wilson, 2016;Chen et al., 2018). Fan et al. (Fan et al., 2020) examined the relationship between the anticancer effect of CUR and CD44 in CRC. Following treatment with CUR, the expression of CD44 was downregulated in HCT-116 and HCT-8 cell lines in a dosedependent manner. In addition, CUR specifically inhibited CD44 + CRC cells. Knockdown of CD44 expression using siRNA led to a significant decrease in the inhibitory effect of CUR against CD44 + CRC cells. Further investigation indicated that the apoptosis of CD44 + CRC cells induced by CUR may be associated with the binding of CUR and the CD44 molecule, which could result in a higher uptake of CUR.

Regulation of heat shock protein 27
Heat shock protein 27 (HSP27), a small heat shock protein, was found to be lowly expressed in normal cells; however, its levels increase following exposure to survival stress (Hou et al., 2016). High expression of HSP27 has been associated with growth, progression, and metastasis of cancer cells (Hung et al., 2017). Liang et al. (2018) demonstrated that silencing of HSP27 inhibited apoptosis and autophagy, consequently inducing resistance to treatment with CUR in CRC cells. They found that DLD-1 cells (with high HSP27 expression) are more sensitive to treatment with CUR than HT-29 cells (with low HSP27 expression). HSP27 knockdown (HSP27-KD) cells presented resistance to CUR, accompanied by decreased levels of antiapoptotic proteins (phospho-Akt, Akt, Bcl-2, and phospho-Bad) and increased levels of apoptotic proteins (Bad and cleaved PARP). A series of CUR-induced effects were abolished in HSP27-KD cells, including apoptosis, G2/M cell cycle arrest, oxidative stress, autophagy, blockage of Akt signaling, and mitochondria-mediated apoptosis. In addition, HSP27-KD cells had decreased levels of antiapoptotic proteins (phospho-Akt, Akt, Bcl-2, and phospho-Bad) and increased levels of apoptotic proteins (Bad and cleaved PARP). These data indicated that HSP27-KD induced resistance to CUR in CRC cells. Chen et al. (2017) demonstrated that treatment with lowdose CUR induced senescence and promoted CDKN1A antisense DNA damage activated RNA (PANDAR) expression in CRC DLD-1 cells. Notably, silencing of Frontiers in Pharmacology frontiersin.org lncRNA PANDAR in these cells may switch senescence to apoptosis partly by regulating the expression of p53upregulated modulator of apoptosis (PUMA). They did not find a difference in the expression of PANDAR between CRC tissues and corresponding normal tissues. Knockdown of PANDAR by si-RNA did not significantly change the proliferation of CRC DLD-1 cells. Moreover, low-dose CUR (5 μM) induced senescence but not apoptosis in DLD-1 cells, along with upregulation of PANDAR expression. Interestingly, silencing of PANDAR in DLD-1 cells treated with CUR was linked to enhanced apoptosis and a significant reduction in senescent cells. They also found that silencing of PANDAR stimulated the mRNA expression of the pro-apoptotic gene PUMA in DLD-1 cells treated with CUR.

Binding to sirtuin 1 and reduction of its stability
Sirtuin 1 (SIRT1) is a NAD+-dependent histone/protein deacetylase with several physiological functions, such as metabolic regulation, differentiation, and stress response . It is involved in the progression of CRC  and highly expressed in human CRC tissues compared with adjacent normal tissues (Song & Surh, 2012). Lee et al. (Lee et al., 2018) found that CUR downregulated the expression of SIRT1 protein, but not mRNA, in human CRC cells . These findings suggested that the regulation of SIRT1 by treatment with CUR is posttranslational.
Frontiers in Pharmacology frontiersin.org  CUR directly bound to cysteine 67 of SIRT1 and resulted in structural modification. Consequently, it promoted the ubiquitin-dependent proteasomal degradation of SIRT1, which is overexpressed in CRC cells. In conclusion, by binding to SIRT1 and decreasing its stability, CUR suppressed its oncogenicity in human CRC cells and inhibited disease progression both in vitro and in vivo.

Inhibition of anchorage-independent growth of colorectal cancer cells
As an in vitro characteristic of tumorigenic cells, anchorageindependent growth (colony-forming capacity in semisolid medium) has been considered a marker of transformed cells which differentiates them from normal cells (Borowicz et al., 2014). In tumor cells, the anchorage-independent growth is related to their tumorigenic and metastatic potential in vivo (Choi et al., 2021). The aberrant epigenetic landscape (i.e., heritable alterations in gene expression without changes in DNA sequence) may increase the complexity of CRC initiation and progression.
Deleted in lung and esophageal cancer 1 (DLEC1), a tumor suppressor gene, could reduce transcriptional activity and promote hypermethylation in several types of cancer, including CRC (Guo Y. et al, 2015). Guo et al. (Guo Y. et al, 2015) investigated the inhibitory role of DLEC1 in anchorageindependent growth of human colorectal adenocarcinoma HT29 cells and epigenetic regulation by CUR. The results revealed the tumor inhibitory role of DLEC1, and indicated its association with the inhibition of anchorage-independent growth of HT29 cells by treatment with CUR. In addition, the investigators demonstrated that CUR suppressed the anchorage-independent growth of HT29 cells by upregulating DLEC1 and reducing CpG methylation in HT29 cells. This activity may be involved in lowering the protein expression of DNMTs and HDACs.
Frontiers in Pharmacology frontiersin.org progression of colitis-associated CRC (Schwabe & Jobin, 2013;McFadden et al., 2015). McFadden et al. (McFadden et al., 2015) used interleukin-10 (IL-10) -/-mice on 129/SvEv background as a model of colitis-associated CRC to evaluate the role of CUR in modulating colonic microbial ecology and preventing the progression of chronic colitis to CRC. Wild-type or IL-10 -/mice (age: 10 weeks) received six weekly intraperitoneal injections of azoxymethane or phosphate-buffered saline, along with supplementation with a control or CUR initiated at the same time. Stools were collected every 4 weeks for microbial community analysis. The results showed that dietary CUR reduced or entirely prevented the development of CRC in the azoxymethane-induced CRC model in a dose-dependent manner. The chemoprophylactic effects appeared to be related to the ecological regulation of colonic microorganisms by CUR, rather than the reduction of inflammation. These findings suggested that the role of CUR in CRC prevention is associated with the maintenance of a more diverse colon microbial ecology.

Conclusion
GIC are associated with high morbidity and mortality rates, thus posing a serious threat to human life and health. Currently, surgery and chemotherapy are the main treatment options for GIC; nevertheless, their efficacy is limited. In numerous studies, CUR, a plant extract with a good safety profile, has exhibited pharmacological effects on GIC both in vivo and in vitro. As demonstrated in the present review, CUR can effectively inhibit GlC through multiple targets, mechanisms, and pathways. Despite the potential of CUR in drug development and application to the treatment of GIC, there remain some deficiencies in the current studies that warrant further investigation.
Regulated cell death (RCD) mainly includes apoptosis, autophagy, pyroptosis, necroptosis, and ferroptosis. Studies investigating RCD induced by CUR in GIC mainly focus on apoptosis and autophagy. It remains unclear whether CUR could induce pyroptosis, necroptosis, and ferroptosis in GIC. These three modes of RCD were recently proposed, and have become hot topics in antitumor drug research, particularly with regard to apoptosis-resistant cells. It has been reported that CUR promoted pyroptosis in liver cancer cells (Li W.-f. et al, 2021), induced necroptosis in prostate and lung cancer cells , and induced ferroptosis in breast  and lung cancer cells (Tang et al., 2021). Further research on the potential induction of pyroptosis, necroptosis, and ferroptosis by CUR in GIC, as well as the mechanisms involved in these processes, is required.
CUR has exhibited good antitumor activity and low toxicity. Nevertheless, its poor solubility in water (456 μg/L) and poor stability in aqueous solution result in an insufficient concentration in serum and tissues, leading to low bioavailability and clinical efficacy (Jakubek et al., 2019). To resolve this problem, researchers have turned their attention to CUR analogs and nanopreparations. This attempt is focused on overcoming its deficiencies in terms of water solubility, bioavailability, and stability, while retaining its excellent antitumor effect. CUR analogs (e.g., WZ35 (He et al., 2019), M37 (Liang et al., 2017), and EF24 (He et al., 2016)) and CUR nanoparticles (e.g., CUR micelles (Lin et al., 2020), CUR-carrying nanoliposomes (Angeline et al., 2020), and solid lipid nanoparticle (Mohamed et al., 2021)) have exerted excellent antitumor effects on GIC. This evidence suggests a breakthrough in the application of CUR to the treatment of GIC and provides broad research prospects.

Author contributions
Conceptualization, YF and JL; writing-original draft preparation,YF; writing-review and editing, YF and XZ; visualization, YT, SC, and JL; funding acquisition, YT. All authors have read and agreed to the published version of the manuscript.

Funding
This work was supported by the National Natural Science Foundation of China (Nos. 81902694) and 345 Talent Project of Shengjing Hospital.


Safety evaluation of the food enzyme pectin lyase from the genetically modified Aspergillus luchuensis strain FLOSC

Abstract The food enzyme pectin lyase ((1→4)‐6‐O‐methyl‐α‐d‐galacturonan lyase; EC 4.2.2.10) is produced with the genetically modified Aspergillus luchuensis (formally Aspergillus niger) strain FLOSC by Advanced Enzyme Technologies Ltd. The genetic modifications do not give rise to safety concerns. The food enzyme is free from viable cells of the production organism and its DNA. The food enzyme is intended to be used in fruit and vegetable processing for juice production. Based on the maximum use level and individual data from the EFSA Comprehensive European Food Database, dietary exposure to the food enzyme–total organic solids (TOS) was estimated to be up to 0.268 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not indicate a safety concern. The systemic toxicity was assessed by means of a repeated dose 90‐day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 794 mg TOS/kg bw per day, the highest dose tested, which when compared with the estimated dietary exposure, results in a margin of exposure of at least 2,900. A search for similarity of the amino acid sequence of the food enzyme to known allergens was made and no match was found. The Panel considered that, under the intended conditions of use the risk of allergic sensitisation and elicitation reactions by dietary exposure cannot be excluded, but the likelihood for this to occur is considered to be low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

'Food enzyme' means a product obtained from plants, animals or micro-organisms or products thereof including a product obtained by a fermentation process using micro-organisms: (i) containing one or more enzymes capable of catalysing a specific biochemical reaction; and (ii) added to food for a technological purpose at any stage of the manufacturing, processing, preparation, treatment, packaging, transport or storage of foods.
'Food enzyme preparation' means a formulation consisting of one or more food enzymes in which substances such as food additives and/or other food ingredients are incorporated to facilitate their storage, sale, standardisation, dilution or dissolution.
Before January 2009, food enzymes other than those used as food additives were not regulated or were regulated as processing aids under the legislation of the Member States. On 20 January 2009, Regulation (EC) No 1332/2008 1 on food enzymes came into force. This Regulation applies to enzymes that are added to food to perform a technological function in the manufacture, processing, preparation, treatment, packaging, transport or storage of such food, including enzymes used as processing aids. Regulation (EC) No 1331/2008 2 established the European Union (EU) procedures for the safety assessment and the authorisation procedure of food additives, food enzymes and food flavourings. The use of a food enzyme shall be authorised only if it is demonstrated that: • it does not pose a safety concern to the health of the consumer at the level of use proposed; • there is a reasonable technological need; • its use does not mislead the consumer.
All food enzymes currently on the European Union market and intended to remain on that market, as well as all new food enzymes, shall be subjected to a safety evaluation by the European Food Safety Authority (EFSA) and approval via an EU Community list.
The 'Guidance on submission of a dossier on food enzymes for safety evaluation' (EFSA CEF Panel, 2009) lays down the administrative, technical and toxicological data required.

1.1.
Background and Terms of Reference as provided by the requestor 1.1.

Background as provided by the European Commission
Only food enzymes included in the European Union (EU) Community list may be placed on the market as such and used in foods, in accordance with the specifications and conditions of use provided for in Article 7 (2) of Regulation (EC) No 1332/2008 1 on food enzymes. Five applications were introduced by the companies 'Novozymes A/S', 'DSM Food Specialties B.V', 'Advanced Enzyme Technologies LtD' and 'the Association of Manufacturers and Formulators of Enzyme Products (AMFEP)' for the authorization of the food enzymes pullulanase from a genetically modified strain of Bacillus subtilis (strain NZYM-AK), glucoamylase from a genetically modified strain of Aspergillus niger (strain NZYM-BW), chymosin from a genetically modified strain of Kluyveromyces lactis (strain CHY), pectin lyase from a genetically modified strain of Aspergillus niger (strain FLOSC) and triacylglycerol lipase from pregastric tissues of cattle, goat and sheep, respectively.
Following the requirements of Article 12. Safety evaluation of the food enzyme pectin lyase from the genetically modified A. luchuensis FLOSC

Terms of Reference
The European Commission requests the European Food Safety Authority to carry out the safety assessments of the food enzymes pullulanase from a genetically modified strain of Bacillus subtilis (strain NZYM-AK), glucoamylase from a genetically modified strain of Aspergillus niger (strain NZYM-BW), chymosin from a genetically modified strain of Kluyveromyces lactis (strain CHY), pectin lyase from a genetically modified strain of Aspergillus niger (strain FLOSC) and triacylglycerol lipase from pregastric tissues of cattle, goat and sheep in accordance with Article 17.3 of Regulation (EC) No 1332/2008 1 on food enzymes.

Interpretation of the Terms of Reference
The present scientific opinion addresses the European Commission's request to carry out the safety assessment of food enzyme pectin lyase from the genetically modified Aspergillus luchuensis strain FLOSC, initially identified as Aspergillus niger.

2.
Data and methodologies

Data
The applicant has submitted a dossier in support of the application for authorisation of the food enzyme pectin lyase from a genetically modified A. niger agg. (strain FLOSC).
Additional information was requested from the applicant during the assessment process on 24 March 2021 and received on 7 June 2021 (see 'Documentation provided to EFSA').

Methodologies
The assessment was conducted in line with the principles described in the EFSA 'Guidance on transparency in the scientific aspects of risk assessment' (EFSA, 2009) and following the relevant guidance documents of EFSA Scientific Committee.
The current Guidance on the submission of a dossier on food enzymes for safety evaluation (EFSA CEF Panel, 2009) as well as the 'Statement on characterisation of microorganisms used for the production of food enzymes' (EFSA CEP Panel, 2019) have been followed for the evaluation of the application with the exception of the exposure assessment, which was carried out in accordance to the updated 'Scientific Guidance for the submission of dossiers on food enzymes' (EFSA CEP Panel, 2021a).

3.
Assessment Pectin lyase catalyses a b-eliminitive cleavage of 1,4-a-D-galactosiduronic linkages in galacturonans, to produce oligosaccharides with 4-deoxy-6-O-methyl-a-D-galact-4-enuronosyl groups at their nonreducing ends. 5 The enzyme is intended to be used in fruit and vegetable processing for juice production. 6
Safety evaluation of the food enzyme pectin lyase from the genetically modified A. luchuensis FLOSC The production strain was identified as A. luchuensis The technical dossier contains all necessary information on the recipient microorganism, the donor organism and the genetic modification process.
The production strain A. luchuensis FLOSC differs from the recipient strain in its capacity to produce the pectin lyase The absence of the antibiotic resistance genes used during the genetic modification was confirmed by PCR analysis.
No issues of concern arising from the genetic modifications were identified by the Panel.

Production of the food enzyme 11
The food enzyme is manufactured according to the Food Hygiene Regulation (EC) No 852/2004 12 , with food safety procedures based on Hazard Analysis and Critical Control Points, and in accordance with current Good Manufacturing Practice. 13 The production strain is grown as a pure culture using a typical industrial medium with conventional process controls in place. Safety evaluation of the food enzyme pectin lyase from the genetically modified A. luchuensis FLOSC Finally, the food enzyme is 14 The applicant provided information on the identity of the substances used to control the fermentation and in the subsequent downstream processing of the food enzyme. 15 The Panel considers that sufficient information has been provided on the manufacturing process and the quality assurance system implemented by the applicant to exclude issues of concern. 3.3.
Characteristics of the food enzyme 3.3.1. Properties of the food enzyme 6 The pectin lyase is a single polypeptide chain of 379 amino acids. 16 The molecular mass of the mature protein, calculated from the amino acid sequence, is 39.8 kDa. 6 The food enzyme was analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis that showed a major protein band corresponding to an apparent molecular mass consistent with the calculated molecular mass of the enzyme, 17 plus several minor bands. A consistent protein pattern was observed across all batches. No other enzymatic activities were reported. 18 The in-house determination of enzyme activity is based on the cleavage of citrus pectin (reaction conditions: pH 5.5, 45°C, 10 min). The enzymatic activity is determined by measuring the release of unsaturated D-4,5 polygalacturonides, which are determined spectrophotometrically at 235 nm. Enzyme activity is expressed in Pectin Lyase Units/g (PLU/g). One unit of pectin lyase (PLU) is defined as the quantity of the enzyme forming one micromole of unsaturated D-4,5 uronide in one minute under the standard assay conditions. 19 The food enzyme has a temperature optimum around 50°C (pH 5.5) and a pH optimum around pH 5.5 (45°C). Thermostability was tested after a pre-incubation of the food enzyme for 120 min at different temperatures (pH 5.5). Enzyme activity decreased above 55°C showing no residual activity above 70°C. 20

Chemical parameters 21
Data on the chemical parameters of the food enzyme were provided for three batches of a dried preparation, one of which (batch 3) was used for the toxicological tests (Table 1). 22 The average total organic solids (TOS) of the three food enzyme batches is 79.2% and the average enzyme activity/mg TOS ratio is 11.9 PLU/mg TOS. Prior to drying the food enzyme is stabilized with 23

Purity 24
The lead content in the three commercial batches was below 0.25 mg/kg 25 which complies with the specification for lead (≤ 5 mg/kg) as laid down in the general specifications and considerations for enzymes used in food processing (FAO/WHO, 2006). In addition, the levels of arsenic, mercury and cadmium were below the limits of detection (LODs) of the employed methodologies. 26,27 The food enzyme complies with the microbiological criteria (for total coliforms, Escherichia coli and Salmonella) as laid down in the general specifications and considerations for enzymes used in food processing (FAO/WHO, 2006). 28 No antimicrobial activity was detected in any of the tested batches. 29 Strains of Aspergillus, in common with most filamentous fungi, have the capacity to produce a range of secondary metabolites (Frisvad et al., 2018). The presence of aflatoxins B1, B2, G1, G2 and M1, ochratoxin A, fumonisin B1, zearalenone, deoxynivalenol, T-2 toxin, HT2-toxin, ergocornine, ergocristine, ergocryptine, ergometrine, ergosine, ergotamine was examined in the three food enzyme batches. All were below the LOD of the applied methods. 30,31 Adverse effects derived from the possible presence of other secondary metabolites is addressed by the toxicological examination of the food enzyme TOS.
The Panel considered that the information provided on the purity of the food enzyme is sufficient.

Viable cells and DNA of the production strain 32
The absence of viable cells of the production strain in the food enzyme was demonstrated in three independent batches analysed in triplicate. 33 No colonies were produced. The absence of recombinant DNA in the food enzyme was demonstrated by PCR analysis of three batches in triplicate. No DNA was detected with with a limit of detection of 10 ng spiked DNA/g food enzyme. 34

Toxicological data 35
A battery of toxicological tests including a bacterial gene mutation assay (Ames test), an in vitro mammalian chromosomal aberration test, and a repeated dose 90-day oral toxicity study in rats has been provided. The toxicological assays were performed with batch 3 (Table 1) which was produced according to the procedure used for commercial batches and is considered suitable as a test item.
The Panel concluded that the food enzyme pectin lyase did not induce gene mutations under the test conditions applied in this study.

In vitro mammalian chromosomal aberration test
The in vitro mammalian chromosomal aberration test was carried out according to OECD Test Guideline 473 (OECD, 2014) and following GLP. 37 An experiment was performed with duplicate cultures of human peripheral blood lymphocytes. The cell cultures were treated with the food enzyme either with or without metabolic activation (S9-mix). The range-finding study was performed at concentrations ranging from 312 to 5,000 lg/mL, and no inhibition of mitotic activity above 50% was seen. Based on these results, cells were exposed to the food enzyme and scored for chromosomal aberrations at concentrations of 1,250, 2,500 and 5,000 lg/mL, corresponding to 993, 1,985 and 3,970 lg TOS/mL, in a short-term treatment (4 h exposure and 20 h recovery period) either with and without S9-mix, and in the continuous treatment (24 h) in the absence of S9-mix. No cytotoxicity was seen either in the short-term (with or without S9-mix) or in the long-term treatment. The frequency of structural and numerical aberrations was not statistically significantly different to the negative controls at any concentration tested.
The Panel concluded that food enzyme pectin lyase did not induce increase in the frequency of structural and numerical aberrations under the test conditions applied in this study.

Repeated dose 90-day oral toxicity study in rodents
The repeated dose 90-day oral toxicity study was performed in accordance with OECD Test Guideline 408 (OECD, 1998) and following GLP. 38 Groups of 10 male and 10 female Sprague-Dawley rats received by gavage the food enzyme in doses of 250, 500 and 1,000 mg/kg bw per day, corresponding to 198.5, 396 and 794 mg TOS/kg bw per day. Controls received the vehicle (distilled water).
Furthermore, a recovery control and a high-dose groups were included in the study, each comprising 6 males and 6 females, and terminated 4 weeks after the end of treatment.
No mortality was observed.
In the functional observations, a statistically significant increase in motor activity (+42.5%) in middose males and a statistically significant decrease (À48.2%) in male recovery group for the first interval were observed. In the female recovery group, a statistically significant increase in motor activity was observed for the first and second interval (+87.4% and +55.9%, respectively). The Panel considered these changes as incidental.
Haematological investigation revealed a statistically significant decrease in mean corpuscular haemoglobin concentration (MCHC) (À1%) and platelets (À20%) in low-dose males. A statistically significant increase in mean corpuscular volume (MCV) in low-, mid-and high-dose females (+4%, +2%, +4%), and a statistically significant decrease in total red blood cells (RBC, À11%) in high-dose females were reported. At the end of the recovery period, a statistically significant decrease in platelets (À15%) in the male recovery group was noted. The Panel considered all these changes as not toxicologically relevant as they were only observed in one sex (all parameters), in absence of a dose-response relationship (MCHC, platelets, MCV), because the changes were low (MCHC, platelets, MCV) and the change was only observed at the end of recovery (platelets).
The Panel considered all these changes as not toxicologically relevant as they were only observed in one sex (ALT, AST, GGT, bilirubin, sodium), in the absence of a dose-response relationship (total protein, ALT, globulin, calcium, phosphorus, sodium, chloride) and because of a low magnitude of the changes (ALT, AST, GGT, total proteins, globulin, calcium, sodium, chloride).
Statistically significant changes in organ weights included an increase in relative liver weight in lowand mid-dose females (+12%, +13%), relative thymus weight in low-, mid-and high-dose females (+46%, +29%, +39%), and a decrease in relative weights of ovaries (À17%) and uterus (À20%) in high-dose female group. At the end of the recovery period, there was a statistically significant increase in relative spleen weight (+14%) in male recovery group and in relative weight of ovaries (+29%) and heart (+27%) in female recovery group. In addition, in female recovery group a statistically significant decrease in relative liver (À18%) and kidney weights (À15%) in comparison to the recovery control group was recorded. The Panel considered the changes in organ weights as not toxicologically relevant as they were only observed in one sex (relative weights of the liver, thymus, heart, kidney, spleen), in the absence of a dose-response relationship (relative weights of the liver and the thymus), no consistency between the direction of the changes at the end of the treatment and after the recovery period (the relative weight of ovaries), only recorded at the end of the recovery period (spleen), a low magnitude of the change (the relative liver weight), no statistically significant changes in the absolute weights and no gross pathological and histopathological changes in organs and tissues were observed.
No other statistically significant or biologically relevant differences to controls were reported. The Panel identified a no observed adverse effect level (NOAEL) of 794 mg TOS/kg bw per day, the highest dose tested.

Allergenicity 39
The allergenicity assessment considers only the food enzyme and not any carrier or other excipient, which may be used in the final formulation.
The potential allergenicity of the pectin lyase produced with the genetically modified A. luchuensis strain FLOSC was assessed by comparing its amino acid sequence with those of known allergens according to the 'Scientific opinion on the assessment of allergenicity of GM plants and microorganisms and derived food and feed of the Scientific Panel on Genetically Modified Organisms' (EFSA GMO Panel, 2010). Using higher than 35% identity in a sliding window of 80 amino acids as the criterion, no match was found. 40 No information is available on oral and respiratory sensitisation or elicitation reactions of this pectin lyase.
Pectate lyases present in plant tissues/or pollen are reported for their role in allergenicity, including the oral allergy syndrome. In Mediterranean areas, Cupressus sempervirens (Italian cypress or Mediterranean cypress) is the most common pollinating species. Pectate lyase from the cypress tree was identified as a group 1 major allergen (Charpin et al., 2019). No matches were found of the enzyme under assessment and lyases from pollen. A link to the oral allergy syndrome based on ingestion of allergens cross-reacting with pollen is not likely.
According to the information provided, substances or products that may cause allergies or intolerances (Regulation ( Safety evaluation of the food enzyme pectin lyase from the genetically modified A. luchuensis FLOSC the microorganisms. However, during the fermentation process, these products will be degraded and utilised by the microorganisms for cell growth, cell maintenance and production of enzyme protein. In addition, the fungal biomass and fermentation solids are removed. Taking into account the fermentation process and downstream processing, the Panel considers that potentially allergenic residues of these materials employed as protein sources are not expected to be present in the food enzyme.
The Panel considers that, under the intended conditions of use, the risk of allergic sensitisation and elicitation reactions upon dietary exposure to this food enzyme cannot be excluded, but the likelihood of such reactions occurring is considered to be low.

3.5.
Dietary exposure 3.5.1. Intended use of the food enzyme The food enzyme is intended to be used in fruit and vegetable processing for juice production 42 at the recommended use levels up to 6.25 mg TOS/kg fruit or vegetable mash. 43 In fruit and vegetable processing for juice production, the food enzyme is added to a mash of fruits or vegetables (with or without peels) 44 , where the pectin lyase cleaves galacturonan-rich cell wall components (e.g. pectin, in particular, highly esterified pectin) to facilitate the release of juice. The enzymatic treatment can lead to higher yields. By using this food enzyme several types of juices can by produced, ready to drink, concentrated and dehydrated juices. 42 The food enzyme remains in the processed juices. The survival of the activity will depend on the food process conditions. 45

Dietary exposure estimation
Chronic exposure was calculated by combining the maximum recommended use level provided by the applicant with the individual data from the EFSA Comprehensive European Food Consumption Database. The estimation involved selection of relevant food categories and application of technical conversion factors (EFSA CEP Panel, 2021b). Exposure from individual FoodEx categories was subsequently summed up, averaged over the total survey period and normalised for body weight. This was done for all individuals across all surveys, resulting in distributions of individual average exposure. Based on these distributions, the mean and 95th percentile exposures were calculated per survey for the total population and per age class. Surveys with only one day per subject were excluded and highlevel exposure/intake was calculated for only those population groups in which the sample size was sufficiently large to allow calculation of the 95th percentile (EFSA, 2011). Table 2 provides an overview of the derived exposure estimates across all surveys. Detailed average and 95th percentile exposure to the food enzyme-TOS per age class, country and survey, as well as contribution from each FoodEx category to the total dietary exposure are reported in Appendix A -Tables 1 and 2. For the present assessment, food consumption data were available from 41 different dietary surveys (covering infants, toddlers, children, adolescents, adults and the elderly), carried out in 22 European countries (Appendix B). The highest dietary exposure at the 95th percentile to the food enzyme-TOS was estimated to be 0.268 mg TOS/kg bw per day in children 3-9 years of age.

Uncertainty analysis
In accordance with the guidance provided in the EFSA opinion related to uncertainties in dietary exposure assessment (EFSA, 2006), the following sources of uncertainties have been considered and are summarised in Table 3.
The conservative approach applied to the exposure estimate to food enzyme-TOS, in particular assumptions made on the occurrence and use levels of this specific food enzyme, is likely to have led to overestimation of the exposure.

3.6.
Margin of exposure A comparison of the NOAEL (794 mg TOS/kg bw per day) from the 90-day rat study with the derived exposure estimates of 0-0.151 mg TOS/kg bw per day at the mean and from 0 to 0.268 mg TOS/kg bw per day at the 95th percentile, resulted in margin of exposure (MOE) of at least 2,963.

Conclusions
Based on the data provided, and the derived margin of exposure, the Panel concluded that the food enzyme pectin lyase produced with a genetically modified strain of A. luchuensis strain FLOSC does not give rise to safety concerns under the intended conditions of use.
The CEP Panel considers the food enzyme free from viable cells of the production organism and recombinant DNA.

Model assumptions and factors
FoodEx categories included in the exposure assessment were assumed to always contain the food enzyme-TOS + Exposure to food enzyme-TOS was always calculated based on the recommended maximum use level + Selection of broad FoodEx categories for the exposure assessment + Use of recipe fractions in disaggregation FoodEx categories +/-Use of technical factors in the exposure model +/-TOS: total organic solids. +: uncertainty with potential to cause overestimation of exposure.
-: uncertainty with potential to cause underestimation of exposure. Safety evaluation of the food enzyme pectin lyase from the genetically modified A. luchuensis FLOSC