Sorafenib is an established therapeutic agent for renal and hepatocellular carcinoma, as well as certain hematologic malignancies, including acute myeloid leukemia (AML). However, prolonged administration of sorafenib can induce drug resistance in cancer cells, making them less responsive to its therapeutic effects. Our findings indicate that sorafenib, dasatinib, and their combination with polymeric micelles (PCL) significantly upregulate pro-apoptotic genes such as Bax and Bad, while downregulating anti-apoptotic genes (BCL-2) and anti-ferroptotic genes (GPX4, NRF2, and SLC7A11), ultimately leading to increased cancer cell death. Furthermore, these treatments enhance apoptosis, elevate levels of reactive oxygen species (ROS) and cellular oxidants, reduce GPX4 activity, diminish colony formation, lower intracellular antioxidant capacity, and concurrently activate both ferroptosis and apoptosis pathways. Resistance to sorafenib can arise in cancer cells due to alterations in the expression of anti-ferroptotic and pro-apoptotic genes, which diminishes the drug’s efficacy over time. Research has shown that the upregulation of the NRF2 gene, resulting from changes in its interaction with KEAP1, leads to the increased expression of anti-ferroptotic genes such as SLC7A11 and GPX4, thereby contributing to sorafenib resistance and the evasion of ferroptosis. Modifying treatment regimens and incorporating second-line agents like dasatinib may provide a strategy to overcome sorafenib resistance; however, these agents carry risks of adverse effects that could lead to secondary resistance and potential harm. Currently, the use of nanocarriers represents a promising strategy to address drug resistance and reduce the necessary dosage of therapeutic agents. Our results demonstrate that the application of nanocarriers in sorafenib and dasatinib nano-systems effectively mitigates sorafenib resistance, resulting in the apoptotic death of MULV-4 cancer cells at significantly lower concentrations.
Toxicity assay of this drugs on MULV-4 sorafenib resistance cells, shown that cells which develop resistance to sorafenib exhibit insensitivity to the drug due to the acquisition of resistance mechanisms and changes in genomic expression profiles. In this study, the utilization of a nano-complex formulation of sorafenib, in combination with the co-administration of dasatinib, has demonstrated significant efficacy in inducing apoptosis in cancer cells. The findings indicate that the sorafenib-dasatinib nanocomplex displays enhanced effectiveness against sorafenib-resistant cells, as evidenced by an IC50 value of 20 nM for the PCL-sorafenib/dasatinib formulation in the sorafenib-resistant MULV-4 cell line.
Gene expression analysis using quantitative reverse transcription polymerase chain reaction (qRT-PCR) revealed a consistent pattern across all treatment groups, characterized by the downregulation of GPX4, SLC7A11, NRF2, and BCL2 genes, alongside the upregulation of BAX, BAD, Caspase-3 (Cas3), and Caspase-8 (Cas8). Notably, the group treated with sorafenib and dasatinib exhibited the most significant changes in the expression levels of the Cas3 and Cas8 genes, whereas the PCL-sorafenib/dasatinib treatment group demonstrated the most pronounced alterations in the expression of all genes examined, with the exception of Cas3 and Cas8.
Consistent with findings from [Author Name]'s study, the results demonstrate that sorafenib, dasatinib, and their respective nanocarriers possess the capability to induce cell cycle arrest by impeding the MAPK signaling pathway during the G1 phase, thereby inhibits cell proliferation(9). The precise mechanism underlying the inhibitory effects of sorafenib and dasatinib on the cell cycle remains elusive; however, prior research has indicated a substantial reduction in the expression of NRAS and KRAS genes following sorafenib treatment, suggesting the suppression of cell signaling pathways and the cessation of cancer cell proliferation in the G1 phase(10). The downregulation of signaling molecules like RAS disrupts signal transduction within the cytosol, ultimately leading to cellular demise.
Upon internalization into the cell, sorafenib and dasatinib elicit an elevation in cytosolic oxidative stress levels, leading to cell death through the modulation of cellular antioxidant pathways. The findings indicate that sorafenib and dasatinib enhance reactive oxygen species (ROS) levels and oxidative stress within the cytosol by impeding the expression and functionality of GPX4(11, 12). Specifically, sorafenib demonstrates a notable reduction in GPX activity; however, the precise mechanism by which this decrease occur whether through downregulation of GPX4 gene expression or direct enzyme inhibition by sorafenib and dasatinib remains unclear. Results from ROS and GPX4 activity assays reveal a twofold increase and a 1.7-fold decrease, respectively, in the NSD treatment group, underscoring the impact of the combined treatment on oxidative stress and GPX4 activity levels.
The results from colony formation assay (CFA) showed that colony size and count were diminished in the treated cells. Similarly to our results a recent study showed significant reduction in the self-renewal capacity of both MCF7 and MDA-MB-231 cells following sorafenib treatment (10, 13). However, Dasatinib, Nano-Sorafenib treated cell, no significant decrease in colony numbers was observed. This lack of impact is attributed to the reduction in the effective dose of sorafenib and dasatinib, which diminished its effect on the colonization of treated cells. The study's key findings show that PCL-Sorafenib-Dasatinib had the most significant impact on colony numbers.
In conclusion, the study elucidates the intricate mechanisms underlying the development of drug resistance to sorafenib in MULV-4 cells and highlights the potential of combined treatments involving sorafenib, dasatinib, and their nano-formulations to overcome resistance. The orchestrated modulation of gene expression, with upregulation of pro-apoptotic genes and downregulation of anti-apoptotic and anti-ferroptotic genes, leads to enhanced apoptosis and disruption of cellular antioxidant pathways. These treatments induce cell death by altering ROS levels, diminishing GPX4 activity, and inhibiting colony formation, ultimately triggering ferroptosis and apoptosis pathways. The findings underscore the promise of nanocarriers in enhancing the efficacy of sorafenib and dasatinib against resistant cancer cells, offering insights into novel therapeutic strategies to combat drug resistance in malignancies.