## 4 Discussion
Sulforaphane induces growth inhibition in breast cancer cells such as MCF-7 in a time and concentration-dependent manner, along with alterations in cell cycle regulatory molecules such as an increase in p21 and p27 and a decrease in cyclin A, cyclin B1, and CDC2 proteins [46]. In this regard, this study confirmed the influence of broccoli on p53 and p21 expression levels and found that NB, broccoli cultivated with DSWM, significantly increased p53 and p21 mRNA expression levels in HT-29 cells. Apoptosis is a type of programmed cell death observed in multicellular organisms. Apoptosis is characterized by morphological and biochemical changes, such as nuclear membrane blebbing, mitochondrial membrane permeability, and caspase cascade activation [47]. Cell apoptosis is primarily mediated by the extrinsic apoptotic pathway involving death receptors and the intrinsic mitochondrial pathway involving multiple caspase family members [48]. A primary mechanism of cell apoptosis is the mitochondrial pathway, which is triggered by the Bcl-2 family and promotes mitochondrial outer membrane permeabilization (MOMP). Pro-apoptotic factors that influence cytochrome C release from mitochondria and anti-apoptotic factors that inhibit cytochrome C release combine to induce apoptosis [49]. Members of the caspase family play essential roles in the initiation and execution of cell death, and these caspases, which are activated in response to signals, are classified into initiator caspases and executioner caspases, influencing the stages of cell apoptosis [50]. Sulforaphane, a major compound in broccoli, was found to induce mitochondria-related cell death and increase the Bax/Bcl-2 ratio in human colorectal cancer cells, SW480. It also activated caspase-3 and caspase-9. Moreover, sulforaphane exhibited growth-inhibitory effects on HT-29 and HCT-116 cells. It was revealed that sulforaphane induced cell cycle arrest at the G2/M phase and apoptosis by upregulating phosphorylation of CDK1 and CDC25B, along with activation of the p38 and JNK pathways [46]. In a study by [PERSON] and [PERSON] [51], it was found that using DSW to improve the quality of green tea significantly inhibited N-nitrosation, which can cause mutagenesis and cellular damage responses. Another study by [PERSON] et al. [14] revealed that the ability of breast cancer cell migration could be inhibited. In this study, treatment with NB, organically cultivated broccoli with DSWM, significantly increased the mRNA expression of cell death factors Bax, Bad, Bim, and Bak in HT-29 cells, as well as the mRNA and protein expression of caspase-9 and caspase-3. Furthermore, the anti-apoptotic factors Bcl-2 and Bcl-xL were less expressed. In this study, the effect of broccoli samples on the production of the inflammatory mediator NO was investigated to evaluate the anti-inflammatory effects. NO plays a vital role in normal physiological processes, such as neurotransmission, vasodilation, and immune defense, but excessive NO production caused by increased iNOS expression promotes inflammatory responses and increases oxidative stress and tissue damage [52]. The exact mechanism by which simple NO production causes disease pathogenesis is unknown, although NO has the chemical potential to cause DNA damage via nitration, nitrosation, and oxidation [53]. In this study, CB, OB, and NB inhibited LPS-induced NO production in RAW 264.7 cells and spleen cells, and the expression levels in NB were most similar to those in the CON group. Macrophages are activated during an inflammatory response to enhance immune defenses, producing several cytokine types. TNF-\(\alpha\), IL-6, IL-18, and IL-12 are examples of pro-inflammatory cytokines produced [54]. These components are required to initiate and improve the inflammatory response, and their expression is increased in LPS-stimulated macrophages [55]. LPS activates M1 macrophages and can also accelerate the inflammatory response by activating or increasing the expression of other pro-inflammatory cytokines and mediators [56].