The findings of the current study shown that, in the colon cancer HT-29 cell line, bFGF alone can activate the expression of anti-apoptotic genes, create a slight increase in survival, and inhibit genes that are effective in triggering apoptosis. In this aspect, bFGF treatment led to a small increase in the metabolic activity of the cells. A small reduction in the ability of cells to form colonies was found when colony formation was examined after bFGF treatment. Previous research on the function of bFGF in the proliferation and/or apoptosis induction of cancer cells has led to some controversial findings. Similar to the findings of our study, Researchers discovered that under apoptotic stress, cells can release bFGF, which in turn promotes the MEK/ERK signaling pathway and increases the production of the anti-apoptotic proteins BCL-2 and MCL-1 [23]. On the other hand, prior research demonstrated that bFGF, via the AKT/MMP-2/VEGF signaling pathway, enhances cell invasion in A549 lung cancer cells [26]. Furthermore, other researches revealed that although morphological alterations caused by bFGF made cells more spherical and fragile, they still maintained metabolic activity and membrane integrity [31, 35, 36]. Accordingly, treatment with bFGF in our study was associated with a reduction in the expression of apoptotic genes like Bax and Caspase 3 and an increase in the expression of anti-apoptotic genes like Bcl-2 and Survivin, as well as an increase in the expression of the Cyclin D1 gene. The cells were also able to sustain their metabolic activity despite the minor morphological modifications. Contrarily, several research have demonstrated that although bFGF upregulates the expression of cyclin D1 in cancer cells via the ERK signaling pathway, it ultimately suppresses the cell cycle in the G2/M phase due to the increase in p21 levels, which slows cell proliferation [35, 36]. In contrast to the results of this study, other studies revealed that bFGF inhibits the Shh pathway in brain tumor cells and reduces cell proliferation by suppressing the expression of Gli-1 and CyclinD1[30]. Furthermore, increased expression of the high molecular weight bFGF isoform through binding to C1QBP induces apoptosis in the HEK293 cell line by activating the ERK1/2 pathway, decreasing mitochondrial Δψm, increasing cytochrome c release, and increasing Caspase 3 cleavage. It can also slow tumor growth in the glioma cell line by interfering with cell cycle progression [27, 35]. According to other findings of previous studies, bFGF causes cell death in TC-32 and TTC-466 cell lines by upregulating the expression of p21 and p53 and downregulating the expression of Bcl-2 [25]. Additionally, raising the level of bFGF in the cytosol using alginate-based nanoparticles (ABNs) coated with bFGF was associated with growth inhibition and cell cycle arrest of Glioma cell lines. It also increased the release of LDH and increased the level of activated ERK1/2 in lung cancer cell line A549 [34]. The researchers demonstrated that the growth inhibitory effect of bFGF at a concentration of 100 µg/ml in the Y1 cell line (murine adrenocortical carcinoma) is dependent on the activation of the Ras-Raf-MAPK pathway as well as the activation of the TGFb signaling pathway, and subsequent DNA damage results in increased expression of p21 which causes cell cycle arrest and cell senescence [33]. On the other hand, bFGF can induce cell death and cell cycle inhibition in Y1 cell line by causing proteotoxic stress and activation of UPR (unfolded protein response) pathway [37]. However, in our study, the first indication consistent with the possibility that bFGF could be able to inhibit the proliferation of cancer cells was seen after a slight rise in apoptotic cells and non-significant decline in the ability of colony formation in the HT-29 cell line. However, it was unable to boost the expression of the genes responsible for the induction of apoptosis.
Contrarily, our results demonstrated that Cyclopamine can cause apoptosis in HT-29 cells. In this study, we used Cyclopamine as a well-known inhibitor of the Shh signalling pathway, which induced apoptosis in colorectal cancer cells. Treatment with cyclopamine was associated with an increase in some of apoptotic genes, inhibition of colon formation, a little decline in metabolic activity of cells, and slight increase in apoptotic cells. According to the results of previous studies, Cyclopamine treatment on the colorectal cancer cell lines alters the morphology of cells, reduces viability, impairs cell migration ability, and eliminates chemotherapy resistance [17]. Additionally, it was discovered that cyclopamine decreases the expression of SMO and GLI1, which inhibits the Shh pathway and may potentially decrease the expression of self-renewal genes in colorectal cancer [16]. Other studies demonstrated that Cyclopamine treatment causes autophagy via the PI3K/AKT pathway in the ovarian cancer cell type by blocking the Shh signaling pathway [19]. Further research has shown that Cyclopamine can contribute in removing chemical resistance to Paclitaxel in the MDA-MB-231 cell line [38]. On the other hand, it has been demonstrated that Cyclopamine Tartrate interferes with mitochondrial function, inhibits cell proliferation, reduces oxygen consumption, increases ROS generation, and induces apoptosis in lung cancer cells [39]. According to the findings of a recent study, cyclopamine had only weakly inhibitive effects on the growth of multiple myeloma cells. However, neither cyclopamine nor circularly polarized trail (CPT) effectively suppressed cell proliferation; rather, the treatment that combined cyclopamine with CPT was successful and restricts the cell proliferation. Therefore, the synchronization impact of apoptosis caused by CPT in cancer cells can be enhanced by Cyclopamine [15]. Similar to these findings, our data show that treatment of HT-29 colorectal cancer cell line with cyclopamine at a concentration of 2.5 µmol/L for 48 hours resulted in increased cells apoptosis, increased expression of the Bax and Caspase 3 genes, as well as decreased colony formation. According to another study, inhibiting the Shh pathway using Cyclopamine, can suppress the growth of kidney cancer cells in the G2/M phase of the cell cycle and decrease the expression of the GLI1, BCL2, VEGFA, and Cyclin D1 genes [40]. In contrast, BCL2 and Cyclin D1 gene expression were not decreased in our study by cyclopamine treatment. Although Cyclin D1 is typically thought of as an oncoprotein, more research should be done on the impact of alterations in its expression in various malignancies [17]. Despite the fact that Cyclin D1 expression has been found to rise in many cancers, some researches have suggested that excessive Cyclin D1 expression may inhibit cell proliferation [33]. For instance, inhibiting the proliferation of breast cancer cells stimulated by bFGF is linked to an upregulation of Cyclin D1. Similarly, Cyclin D1 overexpression in the epithelial HC11 cell line, which resulted in growth inhibition and an increase in apoptosis [27]. Also, treatment with growth inhibitory agents considerably increases the level of cyclin D1 in rat PC12 cells, and cyclin D1 overexpression alone is sufficient to arrest cell growth [23]. These results strongly imply that depending on the cell type, excessive stimulation of cyclin D1 expression by various extracellular stimuli can mediate a growth inhibitory effect [28]. In addition to the mentioned cases, it should be highlighted that, the use of a relatively low dose of cyclopamine in the present study may be to blame for the lack of a decrease in the expression of the BCL2 and cyclin D1 genes.
On the other hand, the findings of our study demonstrated that simultaneous treatment of HT-29 cells with bFGF and cyclopamine triggers apoptosis and is associated to a decrease in anti-apoptotic genes in this cell line. The results also showed a marked decline in the ability to form colonies. According to previous studies, cyclopamine inhibits the Shh pathway by binding to Smoothened, which is upstream of Gli and downstream of Patched. On the other hand, the researchers discovered that after binding to FGFR, bFGF can, in addition to inducing the growth stimulation effect through the Ras/Raf/MEK/ERK signaling pathway, also inhibit Gli through ERK and hence block the Shh pathway. In a 2019 study, Neve A et al. discovered that bFGF at a concentration of 100 ng/mL lowered the expression of GLI1 via activating FGFR and also the ERK pathway in the DAOY Medulloblastoma cell line, and thereby, it influences both cell proliferation and the Shh signaling pathway. Additionally, it was reported in this study that FGFR activation, through inducing differentiation, inhibits proliferation and may ultimately result in cell death in cancer cell lines. The researchers demonstrated that suppressing the Shh pathway by utilizing LDE225 to block SMO led to partial suppression of cell growth in vitro and further inhibition of growth in ex vivo. Moreover, the simultaneous inhibition of SMO and activation of FGFRs by bFGF reduced cell invasion. Also, suppression of FGFRs or inhibition of SMO reduced proliferation in gr3 cells. As a result, the researchers discovered that the impact of FGFR activity on the growth and development of tumors like medulloblastoma is debatable. Instead, it appears that the main impact of the interaction between FGFR and SMO is on the regulation of cell invasion [29]. Similarly, in our study, Cyclopamine's suppression of the SMO and Shh pathways raised the expression of apoptotic genes, decreased the ability to form colonies, and increased apoptotic cells. Furthermore, similar to this study, the present study found that activating the FGFR pathway by bFGF and simultaneously inhibiting the SMO and Shh pathways by Cyclopamine led to a significant reduction in the expression of the anti-apoptotic genes Bcl-2 and Survivin, as well as a reduction in the expression of Cyclin D1 and an increase in cell apoptosis (Fig. 10).