GC is the most common malignancy of the digestive system, with high morbidity and mortality rates worldwide (Johnston and Beckman 2019). Despite developments in treatment over the last few decades, patients with GC still have a poor prognosis (Tan and research 2019, D’Ugo, Agnes et al. 2020). Although cytotoxic chemotherapy remains the cornerstone of treatment for metastatic GC, recent developments in the molecular understanding of the disease have reawakened hope that targeted therapies can be used to improve survival and reduce toxicity (Patel and Cecchini 2020).
NRF2 is a particular transcription factor that strongly regulates antioxidant gene expression. Evidence suggests that malignant tumors, including GC, have higher levels of NRF2 expression and activation, which is linked to enhanced antioxidant capacity, chemoresistance, and a poor clinical prognosis (Farkhondeh, Pourbagher-Shahri et al. 2021). NRF2 has been shown to promote the growth and migration of malignant cells. According to studies, knockdown of NRF2 by specific siRNA reduces the progression of colon cancer (Lee, Kim et al. 2020) and migration of human glioma tumors (Pan, Wang et al. 2013). In this study, we analyzed the combined effect of NRF2 siRNA and paclitaxel on GC cells.
In several malignancies, NRF2 has been found to have a significant impact on cell survival. In this study, we investigated the cytotoxic effect of NRF2 gene suppression on AGS cell survival by MTT assay. The results showed that transfection of cells with NRF2 siRNA leads to a considerable decrease in cell survival compared to the control group. In this way, the results of a study by Lee et al. on SW480 cells from colon cancer showed that inhibition of the NRF2 gene by specific siRNA reduces cell survival and progression (Lee, Kim et al. 2020). Furthermore, Zhang et al. discovered that when the NRF2 gene was knocked down by short hairpin RNA (shRNA) in hepatocellular carcinoma cells the viability of cells was significantly reduced (Zhang, Zhang et al. 2015).
Consistent with MTT findings, annexin-V/PI staining results demonstrated that NRF2 siRNA stimulates apoptosis. However, the increased rate of apoptosis in the combination use of NRF2 siRNA and paclitaxel was more significant. In line with these findings, Bao et al. found that siRNA-mediated NRF2 knockdown promoted cisplatin-induced apoptosis in ovarian carcinoma cells (Bao, Jaramillo et al. 2014). Likewise, another study by Ma et al. discovered that cisplatin treatment and NRF2 gene silencing by shRNA dramatically enhanced the apoptosis rate in cervical cancer as compared to control groups (Ma, Zhang et al. 2012).
To investigate the underlying mechanism, we assessed the expression levels of genes implicated in the apoptotic pathway. The Bcl-2 family comprises the proteins Bcl-2, a pro-apoptotic regulator, Bax, and Caspase-3 and Caspase-9, endogenous apoptotic pathway internal caspases that can regulate cell death via various mechanisms (Shomali, Baradaran et al. 2022, Shomali, Suliman Maashi et al. 2022). The relative mRNA expression of the genes under investigation was assessed by qRT-PCR in control cells, NRF2 siRNA transfected cells, and the combination group. qRT-PCR findings showed that paclitaxel and NRF2 siRNA both upregulated the Bax gene while downregulating the Bcl-2 gene. These alterations in gene expression were more significant when siRNA and paclitaxel were used simultaneously. In Line with our findings, a study by Lee et al. on the SW480 colon cancer cell line found that Bcl-2 expression declines after siRNA-mediated NRF2 knockdown (Lee, Kim et al. 2020). The study by Surikant et al. also showed that NRF2 in lung cancer reduces apoptosis in cancer cells by increasing the expression of Bcl-2 and subsequently enhancing the Bax gene expression (Niture and Jaiswal 2012). The results of qRT-PCR in the current investigation showed that NRF2 siRNA and paclitaxel alone increased the expression of Caspase-3 and Caspase-9 genes. However, this change was more prominent when NRF2 siRNA and paclitaxel were used together. In agreement with our findings, a study by Pan et al. showed that NRF2 suppression by siRNA in the U251 glioblastoma cell line resulted in enhanced apoptosis and upregulated expression and activity of Caspase-3 and − 9 (Pan, Wang et al. 2013). Therefore, it is plausible that NRF2 could inhibit GC cell apoptosis by altering major regulators of apoptosis pathways.
In addition, we assessed cell cycle state in treatment groups to determine the antigrowth effect of NRF2 siRNA and paclitaxel on GC. According to the findings, the number of NRF2-silenced cells was increased during the G1 phase. We also showed that paclitaxel treatment, both alone and in combination with NRF2 siRNA, caused cell cycle arrest in the G2 phase, implying that NRF2 siRNA and paclitaxel could decrease AGS cell proliferation by inducing cell cycle arrest in these phases. In this regard, Homma et al. demonstrated that the knockdown of NRF2 by siRNA arrested the cell cycle at the G1 phase in a human lung cancer cell line (Homma, Ishii et al. 2009). Likewise, Choi et al. investigated the effect of taxol treatment on cell cycle progression in human breast cancer and found that the percentage of G2/M cells was significantly increased compared to untreated controls (Choi and Yoo 2012).
Moreover, the antigrowth effects of NRF2 siRNA and paclitaxel were assessed by measuring the levels of P53 expression in treatment groups. P53 is a transcription factor that is significantly induced by a variety of stress signals, with cell cycle arrest and apoptosis being the most prominent biological effects (Joruiz and Bourdon 2016). According to qRT-PCR analysis, P53 expression was dramatically upregulated in cells transfected with NRF2 siRNA and treated with paclitaxel alone; however, the highest expression of P53 was found in combination therapy. In this way, You et al. performed a study on the link between NRF2 and murine double minute 2 (Mdm2). According to their findings, blocking NRF2 led to the downregulation of Mdm2 in ovarian cancer cells, which then positively regulated P53 signaling and promoted cell death (You, Nam et al. 2011).
Autophagy is an evolutionarily conserved catabolic mechanism that is essential in cancer cell death and survival, as well as cell protection against carcinogenesis (Sui, Chen et al. 2013, Shahverdi, Hajiasgharzadeh et al. 2022). In the current study, we evaluated the impact of NRF2 siRNA in combination with paclitaxel on the induction of autophagy in AGS cells. Our results showed that paclitaxel and NRF2 siRNA transfection enhanced the induction of autophagy compared to the control, suggesting that NRF2 regulates autophagy in GC cells. In this regard, Zhou et al. demonstrated that the silencing of NRF2 enhances baseline autophagy levels in the U251 glioma cell line. Additionally, levels of autophagy were markedly elevated after temozolomide (TMZ) treatment (Zhou, Wang et al. 2013).
One of the most intriguing findings of this research was that suppressing NRF2 could inhibit GC cell migration. The wound healing assay (scratch) results revealed that using either NRF2 siRNA or paclitaxel alone inhibited AGS cell migration. However, when NRF2 siRNA and paclitaxel were combined, migration was more dramatically decreased as compared to the control group, suggesting that NRF2 siRNA and paclitaxel have synergistic anti-metastatic effects on GC cells. In a study by Shen et al., esophageal squamous cell carcinoma cells had enhanced levels of NRF2 expression, and there was a significant protein-level association between NRF2 and hypoxia-inducible factor (HIF)-1α. In addition, the results of the wound healing experiment showed that NRF2 suppression via shRNA reduced HIF-1α expression, hence limiting cell migration (Shen, Yang et al. 2014). Similar findings were reported in research on human glioma cells, which showed that siRNA-mediated NRF2 gene suppression decreased the expression of MMP-9, as well as cell migration (Pan, Wang et al. 2013). Furthermore, Payandeh et al. studied colorectal cancer cells and discovered that siRNA-mediated suppression of NRF2 in combination with Oxaliplatin might dramatically limit cell migration (Payandeh, Tazehkand et al. 2021).
Consequently, we measured MMP-2 expression levels in treatment groups as a metastasis promoter to further examine the mechanism by which NRF2 siRNA may serve as an anti-metastasis agent in GC. According to our findings, MMP-2 was downregulated in cells treated with NRF2 siRNA and paclitaxel, either alone or in combination. Overexpression of MMP-2 has been linked to the depth of invasion and lymph node metastasis of GC (Zheng, Takahashi et al. 2006). In agreement with our findings, Shen et al. found that silencing of NRF2 gene expression by siRNA could significantly downregulate the MMP-2 gene in esophageal squamous cell carcinoma, resulting in decreased cell migration and invasion (Shen, Yang et al. 2014).