Breast cancer remains the cancer most commonly diagnosed in women[1]. Progesterone receptors (PR) and estrogen receptors (ER) belong to the nuclear receptor superfamily. In mammary gland cells, ER/PR deregulation causes tumorigenesis so PR and ER are prognostic markers for breast cancer [1]. Also, ER and PR are predictors of treatment response [2]. Because of developing resistance to endocrine therapy in some patients, most hormonal therapies focus on blocking ER activity and inhibiting aromatase-mediated androgen-to-estrogen conversion [2]. In resistant tumors are need to new therapeutic approaches to provide patients. It may be possible to develop a new treatment strategy for breast cancer by understanding the mechanism of alternative cancer-associated signaling, such as the JAK/STAT signaling.
Tumor cells inhibit apoptosis by decreasing caspase activity, disrupting the balance of pro-apoptotic and anti-apoptotic proteins, and by disrupting death receptor signals [15]. In cancer cells, oncogenic mutations targeting signal transduction pathways and signaling proteins cause loss of proliferation and survival control [16]. Continuous activation of cytoplasmic tyrosine kinases and oncogenic signal transmission accelerates cellular events such as tumor growth, transformation, angiogenesis, and invasion. RTKs regulate signaling pathways such as JAK/STAT. This pathway has a crucial role in proliferation, angiogenesis, and metastasis. Ruxolitinib is an orally available receptor tyrosine kinase inhibitor that targets JAK1 and JAK2. In patients with myelofibrosis or polycythemia vera who are intolerant or inadequately responsive to hydroxyurea, it is approved for use. The most commonly observed toxicities are leukopenia, anemia, thrombocytopenia, headache, bruising, and dizziness [4]. Studies have revealed the need to minimize the side effects of Ruxolitinib and increase drug effectiveness because of the high number of side effects that Ruxolitinib causes on cells and patients [17]. At this point, it is thought that this deficiency can be decreased by synthesizing the nanoform of the drug with nanoparticles, which can directly affect cancer cells more effectively, and selectively increase drug accumulation in tumor cells to reduce the toxic and side effects of drugs.
Due to the biodegradable and biocompatible features of PCL, it is frequently used in drug development studies as it facilitates drug release and long-term degradation up to several months [8]. In literature, have been found studies related with Paclitaxel [12], Quercetin[18], Irinotecan [19], 5-Fluorouracil[20] and Atorvastatin calcium[21] loaded PCL nanoparticle. In the literature, there are just only two studies on the nanoform of ruxolitinib: gold-coated nanoparticles of Ruxolitinib[17] and a topical emulgel containing Ruxolitinib nanoliposome [22]. In this study, Ruxolitinib-loaded PCL nanoparticles were synthesized for the first time in the literature and their effects on apoptotic and JAT/STAT signaling pathways in triple-positive breast cancer cells were examined.
Particle size has an important role in the interaction between NPs and the penetration, cell membrane, and cellular uptake as well as determining the route of treatment. Especially in intravenous applications, the nanoparticle diameter should be below a certain size as it may obstruct blood capillaries. In contrast, smaller particles may have toxic effects because of their greater surface area [23]. Unal et al. [12] showed that the average size of the Paclitaxel-loaded PCL nanoparticles they synthesized was between 199–383 nm and that the nanoparticle size may increase compared to blank nanoparticles due to Paclitaxel settling on the nanoparticle surface. Similarly, in this study, the average sizes of Ruxolitinib, PCL-NP, and Rux-PCL-NP were found to be 564.4641 ± 25.4505 nm, 159.4 ± 74.72 nm, 219 ± 88.66 nm, respectively (Table 1.). Studies have shown that drug-loaded nanoparticles can be larger than empty nanoparticles, and active transport makes carrier systems smaller than 500 nm more effective at delivering drugs to target tissues, especially in cancer research [18]. According to our results, the particle size of ruxolitinib-loaded PCL nanoparticles may have increased compared to PCL-NPs due to the drugs settling on the nanoparticle surface.
The zeta potential plays an important role in determining the characteristics and stability of particles. Studies have shown that positively charged NPs have a high probability of interacting with the cell membrane because the cell membrane surface is negatively charged [24].Yue et al. demonstrated that the positive charge further increased the uptake of NPs into the cell [25]. Unal et al. determined that the interaction between NPs and mucus layer was significantly increased by coating NPs with positively charged materials [26]. PCL has a negative surface charge due to its terminal carboxylic groups. Unal et al. determined that in uncoated NPs, zeta potentials ranged from − 20.1 to -25.8, whereas in coated NPs, surface charges ranged from + 29.6 to + 57. [12]. They demonstrated that the zeta potential of NPs coated with chitosan and PCL used in the study changed from negative to positive [12]. According to our results, the surface charge of Rux-PCL-NPs increased from − 44,6 to 0.471 mV (Table 1.).
Formulation homogeneity is indicated by PDI values. 0 indicates a monodisperse system; 1 indicates a heterogeneous system containing aggregates, polymer residues, and particles of varying sizes. Unal et al. demonstrated that the PDI values of Paclitaxel-loaded PCL and chitosan nanoparticles were close to 0. According to our findings, synthesized nanoparticles are homogeneous since the PDI value of Rux-PCL-NPs is close to 0 (Table 1.).
Studies have shown that the encapsulation efficiency of NPs prepared with high molecular weight (MW) PCL is higher than that of NPs coated with low molecular weight PCL [24, 27]. Increasing organic phase viscosity may lead to higher encapsulation efficiency. Unal et al. demonstrated that the encapsulation efficiency of NPs prepared with 80,000 MW PCL ranged from 59.4–64.7%, while NPs prepared with 14,000 MW PCL varied from 51.3–63.1%. In the same study, they determined that the drug loading capacity of NPs prepared with 80,000 MW PCL was higher compared to those prepared with 14,000 MW PCL, with a range of 6.2–8.4% and 5.2–6.6%, respectively. This suggests that the molecular weight of PCL can influence the amount of drug that can be loaded into the nanoparticles [12]. According to our findings, the %EE values of Rux-PCL-NPs prepared with 66000 MW PCL were found similarly to be 61% and 6.1%, respectively (Table 1.). According to our results, Ruxolitinib coated with PCL may be effective due to the increase in encapsulation values.
Unal et al. determined that the sizes of Paclitaxel-loaded NPs prepared with 80000 MW PCL were 238–380 nm and that the NP formulations were smooth and spherical [12]. According to our findings, the molecular size of Ruxolitinib decreased as a result of its encapsulation with PCL and the size of Rux-PCL-NPs varies between 97–101 nm, and all NP formulations have smooth and spherical surfaces (Figs. 1a, 1b).
Unal et al. demonstrated the in vitro release profiles of PCX from PCL-NPs by the dialysis membrane technique. They determined that Paclitaxel has a release profile from the PCL formulation for up to 96 hours. They showed that a sudden release of Paclitaxel from Paclitaxel-loaded Chitosan (CS) and Paclitaxel-loaded Poly-l-lysine (PLL) nanoparticles occurred in the first 24 hours and the release rate of Paclitaxel may vary depending on the coating material [12]. In the same study, when looking at the rates of drug release according to particle sizes, they found that smaller-sized Paclitaxel-loaded PCL nanoparticles showed a higher release rate and larger-sized Paclitaxel-loaded chitosan-coated PCL nanoparticles showed a slower release [12]. Kamaraj et al. determined that the release of DDA from 14-deoxy 11,12-didehydro andrographolide loaded polycaprolactone nanoparticles (nanoDDA) was 20% in the first 24 hours, increased up to 50% up to 192 hours, and decreased at the 264th hour [28]. According to our findings, Ruxolitinib was released from PCL nanoparticles at a rate of 51% in the first 24 hours, release of ruxolitinib was 77% at the end of the 96th hour. According to our results, a controlled release of Ruxolitinib active ingredients from PCL nanoparticles can be achieved (Fig. 2).
Schneider et al. determined that Ruxolitinib decreases proliferation in SKBR3, MCF-7, and MDAMB-468 cells and the IC50 values of Ruxolitinib are 13.94 µM, 30.42 µM, and 10.87 µM, respectively [29]. Abamor et al. determined that the toxic effects of blank nanoparticles and Quercetin-loaded-PCL nanoparticles were quite low. They demonstrated that even at low concentrations, nanoparticles show similar cell viability to control [18]. Ozturk et al. demonstrated that lower cell viability was obtained with 5-FU-loaded-PCL NPs and empty PCL nanoparticles have no effect on the viability of Caco-2 cells [20]. According to our results, PCL nanoparticles don’t affect the viability of cells (Fig. 3c). In addition, our study indicated that the IC50 value of Ruxolitinib decreased from 50 µM to 0.3267 µM with the treatment of the nanoformulation of Ruxolitinib (Fig. 3a,3b). According to our findings, similar results can be obtained with free Ruxolitinib treatment by treating lower dose concentrations with Rux-PCL-NPs treatment in BT474 cells.
Apoptosis is regulated by a series of events [30]. The loss of mitochondrial membrane potential activates the release of Cytochrome C, located between the mitochondrial double membrane, into the cytoplasm, activates the mitochondrial pathway of apoptosis, and stimulates caspase activation [31]. Members of the protease caspase family have important roles in the initiation and execution of apoptosis [32]. Apoptosis is regulated by pro- and anti-apoptotic members of the BCL-2 family [14]. It causes increased resistance of several cancer cells to chemotherapy due to the activation of MCL-1, BCL-XL, BCL-2, and BCL-W, which are anti-apoptotic members of the BCL-2 family [33]. Li et al. determined that Ruxolitinib treatment for 48 hours increased the expression of cleaved PARP and Caspase − 9, -8, -3 in SW620 and LS411N colorectal cancer cells [34]. Bragta et al demonstrated that flow cytometry analyses showed that Carboplatin-loaded-poly-(ɛ-caprolactone) nanoparticles (CBDCA-PCL-NP) caused B16F1 melanoma cells to undergo 57.6% apoptosis, and CBDCA-PCL-NPs-Gel caused 80.2% apoptosis [11]. They demonstrated that CBDCA-PCL-NPs-Gel treatment decreased Bcl-2 expression in tumor tissues by 2.5-fold and increased Bax expression by 2.03-fold in tumor tissues and B16F1 cells. Also, they determined that the nanoform of Carboplatin can induce Caspase-8 and Caspase-3-mediated apoptosis in tumor tissues and B16F1 melanoma cells [11]. Bhattacharya demonstrated by annexin V assay that the percentage of apoptotic cells in NCI-H460 cells treated with free Gefitinib, Gefitinib PCL10,000NPs, Gefitinib PCL45,000NPs, and Gefitinib PCL80,000NPs increased 30.78 ± 3.78%, 31.67 ± 3.67%, and 46.78 ± 4.56 %, respectively [35]. According to ur results, treatment of Ruxolitinib and Rux-PCL-NPs in BT474 cells inhibits apoptosis decreasing Caspase-8, Caspase-3, Mcl-1, and Bcl-2 expressions (Fig. 5a,5b,5c,5d).
JAK2 has an essential role in regulating the proliferation and apoptosis of cancer cells by activating STAT3/STAT5 and PI3K/AKT pathways [36]. STAT3 and STAT5 modulate the expression of genes involved in cell growth, angiogenesis, and survival [37, 38]. Therefore, drug resistance and relapse may develop. Hyperactivation of STAT3 and STAT5 is caused by mutations in these genes and is associated with cancer progression in patients [39]. The activation of STAT3 occurs in all classes of breast cancer, but is most common in TNBCs [40]. STAT5 stimulates both terminal differentiation of the mammary gland and survival [40]. STAT5 increases the expression of pro-survival genes for instance Bcl-Xl. STAT5 is constitutively active in hormone-responsive breast tumors [40]. Kim et al. demonstrated that the IL-6/STAT3/ROS pathway may be associated not only with breast cancer progression and inflammation but also with increased formation of breast cancer stem cells [33]. Pro- and anti-apoptotic proteins such as Bak/Bax and Bcl-2 are also associated with the JAK/STAT signaling pathway. Boca et al. demonstrated that Ruxolitinib coated with gold nanoparticles prevented the proliferation of fibroblasts by blocking JAK2 expression in fibroblast cells [17]. No study was found about nanoforms of ruxolitinib with another polymer in the literature. According to our findings, STAT5 and JAK2 expressions decreased with Rux-PCL-NP treatment in BT474 cells, and there was no significant change in STAT3 expression (Fig. 6a,6b).