###### Abstract  
Plants from the family Brassicaceae produce brassinin (BSN), which is an essential indole phytoplankton. BSN can kill certain types of cancer cells. Using hepatocarcinoma (HCC) cells, we examined the molecular mechanisms of BSN. We found that HCC cell growth was suppressed and apoptosis was induced by BSN via the downregulation of the JAK/STAT3 pathway. The cytoplasmic latent transcription factor STAT3, belonging to the STAT family, acted as both a signal transducer and an activator and was linked to tumor progression and decreased survival. BSN incubation caused HCC cells to produce reactive oxygen species (ROS). By activating caspase-9/-3 and PARP cleavage, Bcl-2 was reduced, and apoptosis was increased. BSN inhibited constitutive STAT3, JAK2, and Src phosphorylation. The JAK/STAT signaling cascade was confirmed by siRNA silencing STAT3 in HCC cells. BSN also suppressed apoptosis by Z-Val-Ala-Asp-Fluoromethylketone (Z-VAD-FMK), an apoptotic inhibitor. N-acetylcysteine (NAC) inhibited the production of ROS and diminished BSN-induced apoptosis. Our findings suggested that BSN has potential as a treatment for cancer.  
happocellular carcinoma; brassinin; STAT3; transcription factors; apoptosis; ROS +
Footnote †: journal: Medical  
(EMT) in lung cancer cells and induce apoptosis in prostate cancer cells by modulating PI3K/Akt/mTOR [8]. BSN inhibited PI3K signaling cascades in colon cancer cells and boosted p21 and p27 expression [4].  
Different types of cancers have abnormal STAT3 activation [9; 10; 11; 12; 13]. STAT3 was phosphorylated by JAK1/2 and Src following stimulation by cytokines and growth factors [14]. According to various studies, blocking STAT3 signaling could be an effective way to fight human cancer. Tumor cells can be affected by oxidative stress in multiple ways. Reducing the reactive oxygen species (ROS) levels has been beneficial, but excessively low levels have caused tumors [15]. Radiation, drugs, pollutants, xenobiotics, and other exogenous factors can produce ROS intracellularly. By modulating various cell signaling pathways, ROS has also been shown to control different hallmarks of cancer cells, such as STAT3, protein kinases, growth factors, cytokines, and others [16]. It has been reported that STAT3 activation is deregulated in many human cancers, including HCC, and it is associated with cancer progression, chemoresistance, a poor prognosis, and a shorter lifespan. Deregulating STAT3 cascades has been suggested as a promising therapeutic strategy for countering STAT3's oncogenic and pro-survival effects in human cancers [1].  
In the present study, HepG2 and C3A cells were used that showed distinct differences in energy metabolism and urea production, as compared to primary hepatocytes. Restoring primary hepatocyte characteristics, such as the urea cycle, is crucial. For example, HepG2 cells were successfully transfected with cDNA-encoding urea cycle enzymes to enhance the application of HepG2 and C3A cells for drug testing and bioartificial liver applications [17]. In this study, we also demonstrated the significance of the STAT3 pathway in the growth of HepG2-C3A cells. STAT3 plays a crucial role in several aspects of HCC survival, proliferation, invasion, and angiogenesis. Therefore, we explored whether BSN could provide its anti-proliferative and pro-apoptotic effects in HCC cells by suppressing STAT3 signaling in HCC cells.