Abstract
Background/Aim: The over-expression of P-glycoprotein (P-gp) is a major mechanism underlying multidrug resistance (MDR). Co-treatment with Janus kinase 2 (Jak2) inhibitors sensitizes P-gp-over-expressing drug-resistant cancer cells. In this study, we evaluated pacritinib, a Jak2 inhibitor currently in phase III clinical trials. Materials and Methods: Microscopic observation, cell viability assay, colony forming assay, rhodamine uptake tests, annexin V analyses, fluorescence-activated cell sorting (FACS), and western-blot analysis were performed to further investigate the mechanism of action. Results: We found that pacritinib reduced cell viability, induced G2 arrest, and upregulated early apoptosis when administered to P-gp-over-expressing resistant KBV20C cells with vincristine (VIC). Moreover, apoptosis and G2 arrest in VIC–pacritinib-treated cells were involved in the upregulation of pH2AX expression. Pacritinib had an approximately 2-fold higher P-gp-inhibitory activity than the dimethyl sulfoxide (DMSO)-treated control, indicating that VIC–pacritinib sensitization involves the P-gp-inhibitory effects of pacritinib. Similar to VIC, other antimitotic drugs (vinorelbine, vinblastine, and eribulin) could also sensitize against KBV20C cells by co-treatment with pacritinib. Furthermore, comparison of pacritinib with previously characterized Jak2 inhibitors revealed that the VIC–pacritinib combination had sensitization effects similar to those of VIC– CEP-33779 or VIC–NVP-BSK805 combinations at lower doses in KBV20C cells. Generally, Jak2 inhibitor and VIC co-treatment sensitized P-gp-over-expressing resistant cancer cells by inducing early apoptosis. Conclusion: Collectively, pacritinib, induced G2 arrest, reduced cell viability, had high P-gp inhibitory activity, and upregulated the expression of pH2AX when used in combination with VIC. As pacritinib is a Jak2 inhibitor currently in phase III clinical trials, our findings may facilitate the application of this co-treatment in patients with MDR cancer.
Anti-mitotic drugs, such as vincristine (VIC), eribulin, vinorelbine, and vinblastine, prevent microtubule polymerization or depolymerization (1, 2). As multidrug resistance (MDR) can develop in patients administered these drugs, investigations for sensitizing the underlying mechanisms to antimitotic drugs are important to facilitate the faster and more effective treatment of patients with MDR.
P-glycoprotein (P-gp) is located in the cellular membrane and is responsible for the efflux of anticancer drugs, and its over-expression is a well-known mechanism underlying MDR in cancer cells (3-6). Although many inhibitors with P-gp activity have been examined in clinics, toxicity in normal tissues has prevented the use of these P-gp inhibitors in resistant cancer patients (4, 5, 7). Investigators are continuously trying to develop P-gp inhibitors with reduced toxicity in normal cells that specifically target drug-resistant cancer cells (5, 8). We have previously identified novel mechanisms and inhibitors that target P-gp activity with low toxicity in normal cells; therefore, we are interested in repositioning drugs, known for their toxicity, with FDA approval (9-11). Once we found instances of novel, FDA-approved drug repositioning for sensitizing P-gp-over-expressing resistant cancers, we believed that these drugs may be administered, without further toxicity tests, to MDR cancer patients.
The over-expression of Janus kinase 2 (Jak2) can lead to the development of drug-resistance in cancer cells (12-15). Therefore, treatment with combinations of Jak2 inhibitors has been suggested to overcome MDR (16, 17). In addition, CEP-33779, NVP-BSK805, and XL019 (Jak2 inhibitors) have been shown to increase apoptosis in P-gp-over-expressing antimitotic drug-resistant cancer cells (18-20). As various Jak2 inhibitors have been developed for more effectively treating cancers where Jak2 is over-expressed, we also aimed to identify better Jak2 inhibitors for treating MDR. With the increasing popularity of personalized medicine, we hope that our findings could lead to identifying more specific Jak2 inhibitors for patients with MDR.
In this study, we investigated whether pacritinib, a Jak2 inhibitor currently undergoing phase III clinical trials (21-24), can increase apoptosis in VIC-treated P-gp-over-expressing drug-resistant KBV20C cancer cells. We further investigated the mechanisms underlying the sensitization to VIC−pacritinib co-treatment. Our results can facilitate the development of Jak2 inhibitor-based therapies for drug-resistant cancer patients.
Materials and Methods
Reagents and cell culture. Rhodamine123 (Rhodamine) and verapamil were purchased from Sigma-Aldrich (St. Louis, MO, USA). VIC, vinorelbine, and vinblastine were purchased from Enzo Life Sciences (Farmingdale, NY, USA). Pacritinib, fedratinib, CEP-33779, NVP-BSK805, momelotinib, WP1066, and aripiprazole were purchased from Selleckchem (Houston, TX, USA). Aqueous solutions of eribulin (Eisai Korea, Seoul, Republic of Korea) were obtained from the National Cancer Center in South Korea.
Human oral squamous carcinoma cell line, KB, and its multidrug-resistant subline, KBV20C, were obtained from Dr. Yong Kee Kim (College of Pharmacy, Sookmyung Women’s University, Seoul, Republic of h Korea) and have been previously described (25, 26). Cells were grown in RPMI medium containing 10% fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin (WelGENE, Daegu, Republic of Korea).
Microscopic observation. Microscopic observations were performed as previously described (27-29), in order to examine the effect of pacritinib, verapamil, momelotinib, or WP1066, alone and in combination with vincristine for 24 h. All cells were observed using an inverted microscope at ×40 magnification. All experiments were qualitatively analyzed, and the results were confirmed in at least two independent experiments.
Cell viability assay. Colorimetric assay using the EZ-CyTox cell viability assay kit (Daeillab, South Korea) was used for measuring cellular proliferation, as described previously (30-32). Briefly, cells with 30-40% confluence were grown in wells of 96-well plates. The cells were then treated with 5 -10 nM vincristine, 0.5-2 μM pacritinib, 5 nM vincristine + 0.5 μM pacritinib, or 0.1% dimethyl sulfoxide (DMSO) (Control) for 1 or 2 days. EZ-CyTox solution was then added for 1 h at 37°C. Absorbance at 450 nm was measured using the VERSA MAX Microplate Reader (Molecular Devices Corp., Sunnyvale, CA, USA). All experiments were performed at least in triplicate and repeated twice. The quantitative analysis was performed in at least two independent experiments in triplicate.
Colony forming assay. The colony forming assay was used to assess long-term growth in the presence of a drug based on a previously described method (33). Briefly, 1-2×103 cells were grown in 6-well plates for 5-6 days, and then stimulated with 5 nM vincristine, 0.5 μM pacritinib, 1 μM pacritinib, 5 nM vincristine + 0.5 μM pacritinib, 5 nM vincristine + 1 μM pacritinib, or 0.1% DMSO (Control) for 5-6 days. The fresh medium containing the drugs was changed twice. Colony forming assays were immediately measured using crystal violet staining, after 10-12 days. Viable colonies were fixed with 4% parafomaldehyde, stained with 0.05% crystal violet for 20 min, washed with phosphate buffered saline (PBS), and air-dried. Relative colonies were analyzed using an image analyzer. All performed experiments were repeated at least twice.
Fluorescence-activated cell sorting (FACS) analysis. FACS was performed as previously described (27-29), in order to determine whether pacritinib led to cell-cycle arrest. Briefly, cells with 30-40% confluence were treated with 0.5 μM pacritinib, 1 μM pacritinib, or 0.1% DMSO (Control), alone and in combination with 5 nM vincristine, 5 nM vinblastine, 0.1 μg/ml vinorelbine, or 30 nM eribulin for 24 h. The cells were then treated with trypsin and collected by centrifugation. The pelleted cells were washed, suspended in 75% ethanol for at least 24 h at −20°C, washed with PBS, and suspended in a propidium iodide and RNase A staining solution for 30 min at 37°C. The cell-cycle distribution of stained cells was qualitatively analyzed and the results confirmed in at least two independent experiments using a Guava EasyCyte Plus Flow Cytometer (Merck Millipore, Burlington, MA, USA).
Annexin V analysis. Annexin V analysis was performed using an annexin V-fluorescein isothiocyanate staining kit (BD Bioscience, Franklin, NJ, USA) as previously described (27-29), in order to measure apoptosis quantitatively. Briefly, cells with 30-40% confluence were treated with 0.5 μM pacritinib, 1 μM pacritinib, 10 μM verapamil, 2 μM CEP-33779, 2 μM NVP-BSK805, 2 μM momelotinib, 2 μM WP1066, or 0.1% DMSO (Control), alone and in combination with 5 nM vincristine or 30 nM eribulin for 24 h. The cells were then treated with trypsin and collected by centrifugation. The pelleted cells were washed and re-suspended in PBS. Cells with annexin V-fluorescein isothiocyanate and propidium iodide solution were then incubated for 30 min at 25°C. The stained cells were qualitatively analyzed, and the results were confirmed in at least two independent experiments using a Novocyte Flow Cytometer (ACEA Biosciences, San Diego, CA, USA).
Rhodamine uptake tests. The tests used to assess the ability of a drug to inhibit P-gp were based on a previously described method (28, 30, 34). Briefly, cells grown in 60-mm diameter dishes were treated with 10 μM verapamil, 2.5 μM aripiprazole, 2 μM pacritinib, 1 μM pacritinib, or 0.5 μM pacritinib and then incubated for 1 h at 37°C. The medium was removed, and the cells were washed with PBS. The stained cells for 3 h were analyzed using a Guava EasyCyte Plus Flow Cytometer (Merck Millipore, Burlington, MA, USA). All performed experiments were repeated at least twice.
Western blot analysis. Total cellular proteins were extracted as previously described (28, 34, 35). Briefly, cells were treated with 5 nM vincristine, 0.5 μM pacritinib, 1 μM pacritinib, 5 nM vincristine + 0.5 μM pacritinib, 5 nM vincristine + 1 μM pacritinib, or 0.1% DMSO (Control) for 24 h, and then washed with PBS and detached using trypsin. PRO-PREP™ protein extract solution (iNtRON, Seongnam, Korea) was used for isolating total proteins. A protein assay kit (Bio-Rad, Hercules, CA, USA) was used for measuring protein concentrations. The proteins were then subjected to western blot analysis as previously described (28, 34, 35).
Antibodies against C-PARP and Bcl2 were obtained from Cell Signaling Technology (Danvers, MA, USA). Antibodies against Cyclin D1, CDK2, and GAPDH were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibody against pH2AX was obtained from Sigma-Aldrich (St. Louis, MO, USA). Antibodies against α-LC3B, Bax, and CDK1 were obtained from Abcam (Cambridge, UK).
Statistical analysis. All the data are presented as mean±S.D. from at least two independent experiments with triplicates. Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by Bonferroni’s test. Analysis was performed using Graph Pad Prism Software Version 5.0 (GraphPad Software, CA, USA). A p-value of <0.05 was considered as statistically significant.
Results
Co-treatment with pacritinib increases the sensitization of drug-resistant KBV20C cancer cells to VIC treatment. We aimed to identify repurposed drugs that sensitize drug-resistant cancer cells over-expressing P-gp to chemotherapeutic drugs. Previously, Jak2 inhibitors displayed drug-sensitization effects in P-gp-over-expressing drug-resistant cancer cells (18-20). Therefore, identifying novel Jak2 inhibitors and their mechanisms in resistant cancers is important for broadening their clinical applications. Here, we evaluated a recently developed and phase III Jak2 inhibitor, pacritinib (21-24). We used P-gp-over-expressing drug-resistant KBV20C cells, which showed a VIC-resistant phenotype (Figure 1A). We obtained the IC50 values of 1.6 μM and 1.2 μM after 24 and 48 h of treatment, respectively, in drug-resistant KBV20C cells (Figure 1A). To determine the efficiency of pacritinib in combination with VIC in resistant KBV20C cells, we used 0.5 or 1 μM of pacritinib, which has a lower IC50 for pacritinib.
Co-treatment of VIC with pacritinib specifically increases the sensitization of drug-resistant KBV20C cancer cells with low P-gp inhibitory activity. (A-B) Drug-resistant KBV20C were plated on 96-well plates and grown to 30%-40% confluence. The cells were then stimulated for 24 h or 48 h with 5 nM vincristine (VIC-5), 10 nM vincristine (VIC-10), 0.5 μM pacritinib (PACR-0.5), 1 μM pacritinib (PACR-1), 2 μM pacritinib (PACR-2), 5 nM VIC with 0.5 μM pacritinib (VIC+PACR-0.5), or 0.1% DMSO (CON). Cell viability assay was performed as described in Materials and Methods. The data are presented as the mean±SD of at least two experiments repeated in triplicate. For co-treatments, significantly different at *p<0.05 compared to the corresponding control. (C) Drug-resistant KBV20C were grown on 60 mm-diameter dishes and treated with 5 nM vincristine, 0.5 μM pacritinib (pacritinib-0.5), 1 μM pacritinib (pacritinib-1), 5 nM VIC with 0.5 μM pacritinib (VIC+PACR-0.5), 5 nM VIC with 1 μM pacritinib (VIC+PACR-1), or 0.1% DMSO (Control). After 1 day, all cells were observed using an inverted microscope at ×100 magnification. (D-E) KBV20C cells were grown on 60 mm-diameter dishes and treated with 2.5 μM aripiprazole, 10 μM verapamil, 0.5 μM pacritinib, 1 μM pacritinib, 2 μM pacritinib, 5 μM pacritinib, or 0.1% DMSO (Control). After 1 h, all cells were stained with rhodamine for 3 h and examined by using FACS analysis, as described in Materials and Methods. The data (E) are presented as the mean±SD of three experiments. Results were considered statistically significant compared to those of the control when *p<0.05.
As shown in Figure 1B, co-treatment with pacritinib reduced the proliferation of VIC-treated KBV20C cells when compared to single treatment with either VIC or pacritinib. We confirmed the high sensitization effects of VIC– pacritinib co-treatment in resistant KBV20C cells through microscopic observations (Figure 1C). Collectively, we demonstrated that a low dose of pacritinib can sensitize VIC-treated resistant KBV20C cells.
The phase III Jak2 inhibitor, pacritinib, presents P-gp inhibiting activity. Next, we assessed the P-gp inhibitory activity of pacritinib in P-gp-over-expressing KBV20C cells, because we hypothesized that P-gp inhibition by pacritinib may be responsible for its sensitizing effects in VIC-treated KBV20C cells.
Aripiprazole and verapamil, P-gp inhibitors known to increase the inhibition of P-gp substrate efflux (26, 28, 36), were used as positive controls (28, 36). Rhodamine 123, a well-known P-gp substrate, was used to measure P-gp inhibition (26, 28, 34). In this experiment, yellow fluorescence in the cell was indicative of the intracellular accumulation of rhodamine for 3 h. As seen in Figure 1D and E, we observed approximately four- to six-fold higher P-gp inhibitory activity in the positive controls (aripiprazole and verapamil) than in the DMSO-treated negative control. We also found that pacritinib increased P-gp inhibitory activity (Figure 1D and E), suggesting that P-gp inhibition by pacritinib plays a role in the sensitization effect of VIC– pacritinib co-treatment. However, P-gp inhibition by pacritinib was much less than that by positive controls, which was approximately 2-fold higher than that of the DMSO-treated control (Figure 1D and E). Considering that even with lower P-gp-inhibitory activity, pacritinib still sensitized KBV20C cells to VIC, we assumed that pacritinib could increase sensitization of VIC-treated KBV20C cells via both P-gp inhibition and other stimulating mechanisms. Therefore, it is possible that low-dose treatment with pacritinib might be useful in clinical settings because of its minimal toxic P-gp-inhibitory effects in normal cells.
Co-treatment with VIC and pacritinib reduces long-term survival as determined by colony forming assays. Next, we evaluated whether pacritinib at low doses could sensitize drug-resistant KBV20C cancer cells with long-term survival. We performed colony-forming assays, which can evaluate 10 days after drug treatment. As seen in Figure 2A and B, both 0.5 μM and 1 μM pacritinib reduced colony formation in VIC-treated KBV20C cells, whereas single treatment using either VIC or pacritinib showed similar size and numbers to control levels. These results suggest that combination therapy with pacritinib can be used for chemotherapeutic drug-treated resistant cancer cells with long-term efficacy.
Apoptosis induces G2 arrest in VIC–pacritinib co-treated KBV20C cells. (A-B) KBV20C cells were grown on 6-well plates for 5 days and then stimulated with 5 nM vincristine (VIC), 0.5 μM pacritinib (Pacritinib-0.5), 1 μM pacritinib (pacritinib-1), 5 nM VIC with 0.5 μM pacritinib (VIC+PACR-0.5), 5 nM VIC with 1 μM pacritinib (VIC+PACR-1), or 0.1% DMSO (Control) for 5-6 days. Colony forming assays were immediately measured with crystal violet-staining, after 10-12 days, as described in Materials and Methods. The data (B) are presented as the mean±SD of three experiments. Results were considered statistically significant compared to those of the control when *p<0.05. (C-D) KBV20C cells were grown on 60 mm-diameter dishes and treated with 2.5 nM vincristine (VIC-2.5), 5 nM vincristine (VIC-5), 0.5 μM pacritinib (PACR-0.5), 1 μM pacritinib (PACR-1), 2.5 nM VIC with 0.5 μM pacritinib (VIC-2.5+PACR-0.5), 5 nM VIC with 0.5 μM pacritinib (VIC-5+PACR-0.5), 5 nM VIC with 1 μM pacritinib (VIC-5+PACR-1), or 0.1% DMSO (Control). After 24 h, annexin V analyses were performed as described in Materials and Methods. (E) Drug-resistant KBV20C were grown on 60 mm-diameter dishes and treated with 2.5 nM vincristine (VIC-2.5), 5 nM vincristine (VIC-5), 0.5 μM pacritinib (PACR-0.5), 2.5 nM VIC with 0.5 μM pacritinib (VIC-2.5+PACR-0.5), 5 nM VIC with 0.5 μM pacritinib (VIC-5+PACR-0.5), or 0.1% DMSO (Control). After 1 day, all cells were observed using an inverted microscope at ×40 magnification. (F) KBV20C cells were plated on 60 mm-diameter dishes and treated with 5 nM vincristine (VIC), 0.5 μM pacritinib (PACR-0.5), 1 μM pacritinib (PACR-1), 5 nM VIC with 0.5 μM pacritinib (VIC+PACR-0.5), 5 nM VIC with 1 μM pacritinib (VIC+PACR-1), or 0.1% DMSO (CON). After 24 h, western blot analysis was performed using antibodies against C-PARP, Bax, Bcl2, α-LC3B, and GAPDH.
Co-treatment with VIC and pacritinib induces apoptosis of KBV20C cells in a dose-dependent manner. To further clarify the mechanism of action of VIC–pacritinib co-treatment, apoptotic analysis using annexin V staining was performed. When the proportion of apoptotic cells (in both early and late phases) was quantitatively estimated, we found that the proportion of early apoptotic cells was significantly greater than that of late apoptotic cells in both 0.5 μM and 1 μM pacritinib (Figure 2C). We also observed that 1 μM pacritinib was more efficacious than 0.5 μM pacritinib (Figure 2C), suggesting a dose-dependent relationship between Jak2 inhibitors and increased early apoptosis of antimitotic drug-resistant cells. However, when we compared co-treated pacritinib with either 2.5 nM or 5 nM of VIC, we observed little increase in early apoptosis in 5 nM VIC than 2.5 nM VIC (Figure 2D and E). These results suggest that early apoptosis in VIC–pacritinib co-treated cells can be highly increased with an increase in pacritinib concentration. Collectively, these results indicate that early apoptotic induction results in the sensitization effects of VIC–pacritinib co-treatment.
To confirm increased apoptosis in co-treated cells in the presence of molecular markers, we measured C-PARP production. As shown in Figure 2F, C-PARP expression level increased in VIC–pacritinib-treated cells. When we compared the C-PARP expression levels in cells treated with different concentrations of pacritinib (between 0.5 μM and 1 μM pacritinib), we found that C-PARP expression level increased in a dose-dependent manner in VIC-treated resistant KBV20C cells, whereas its expression level did not change much by single treatment with either pacritinib or VIC. This indicates that pacritinib is highly effective when administered as a combination therapy in patients with resistant cancers. Altogether, we demonstrated that pacritinib can highly sensitize resistant cancers when administered in combination with VIC through the early apoptotic pathway.
Additionally, we assessed apoptosis-related proteins (Bax and Bcl2) and autophagy-related proteins (α-LC3B). As seen in Figure 2F, those proteins were not changed much in VIC−pacritinib co-treated cells, suggesting that VIC– pacritinib co-treatment did not induce autophagic death in drug-resistant KBV20C cells.
VIC–pacritinib co-treatment induces G2-arrest and increases DNA damage in KBV20C cells. To further clarify the mechanism of action of VIC–pacritinib co-treatment, we performed FACS analyses. As shown in Figure 3A, co-treatment with VIC and pacritinib increased the number of cells in G2 arrest compared with that observed in single treatments with either agent. A positive relationship was found between the dose of pacritinib and the proportion of VIC-treated cells in G2 arrest.
Pacritinib increases the sensitization of KBV20C cells treated with other antimitotic drugs. (A-B) KBV20C cells were grown on 60 mm-diameter dishes and treated with 5 nM vincristine, 5 nM vinblastine, 0.1 μg/ml vinorelbine, 30 nM eribulin, 0.5 μM pacritinib (PACR-0.5), 1 μM pacritinib (PACR-1), 5 nM VIC with 0.5 μM pacritinib (VIC+PACR-0.5), 5 nM VIC with 1 μM pacritinib (VIC+PACR-1), 5 nM vinblastine with 1 μM pacritinib (VIB+PACR-1), 0.1 μg/ml vinorelbine with 1μM pacritinib (VIO+PACR), 30 nM eribulin with 1 μM pacritinib (ERI+PACR), or 0.1% DMSO (Control). After 24 h, FACS analyses were performed as described in Materials and Methods. (C) KBV20C cells were grown on 60 mm-diameter dishes and treated with 30 nM eribulin, 1 μM pacritinib (PACR-1), 30 nM eribulin with 1 μM pacritinib (ERI+PACR-1), or 0.1% DMSO (Control). After 24 h, annexin V analyses were performed as described in Materials and Methods. (D) KBV20C cells were plated on 60 mm-diameter dishes and treated with 5 nM vincristine (VIC), 0.5 μM pacritinib (PACR-0.5), 5 nM VIC with 0.5 μM pacritinib (VIC+PACR-0.5), or 0.1% DMSO (CON). After 24 h, western blot analysis was performed using antibodies against Cyclin D1, CDK2, CDK1, pH2AX, and GAPDH.
VIC–pacritinib co-treatment increases the sensitization of KBV20C cells to other antimitotic drugs. We also investigated whether pacritinib was effective in combination with other antimitotic drugs. We assessed the sensitization effect of pacritinib in combination with vinorelbine and vinblastine, which are antimitotic drugs that are routinely used as chemotherapeutic agents in cancer (2, 37). As shown in Figure 3A, vinorelbine-pacritinib and vinblastine– pacritinib highly increased G2 arrest in P-gp-over-expressing resistant KBV20C cells, when compared to single treatment with either vinorelbine or vinblastine.
The KBV20C cell line is a particularly useful model of highly eribulin-resistant cancer cells (26, 38, 39). As shown in Figure 3B, eribulin–pacritinib also increased G2 arrest in P-gp-over-expressing resistant KBV20C cells when compared to single treatment with eribulin. As seen in the annexin V analysis shown in Figure 3C, eribulin–pacritinib produced a greater increase in early apoptosis, which is similar to the VIC– pacritinib results (Figure 2C and D). The results confirmed that eribulin–pacritinib was as effective as VIC–pacritinib in sensitizing drug-resistant KBV20C cancer cells. This also suggests that pacritinib can be used at a low dose to sensitize eribulin-resistant cancer cells. This result suggests that pacritinib could be combined with other antimitotic drugs to sensitize cancer cells over-expressing P-gp. We conclude that pacritinib can be used to treat various drug-resistant cancer patients. To further investigate the expression of proteins involved in G2 arrest (40, 41), we used western blotting analysis. As seen in Figure 3D, the cyclin protein expression in VIC–pacritinib co-treatment was not qualitatively different compared with that observed in single treatment with either agent. We found that the expression level of pH2AX, a DNA damage marker, was significantly increased (Figure 3D), suggesting that DNA damage may increase G2 arrest in VIC–pacritinib-treated KBV20C cells. We conclude that the DNA damage signal increased apoptosis via G2 arrest in VIC–pacritinib-treated KBV20C cells.
Lower pacritinib dose sufficiently sensitizes VIC-treated KBV20C cells similar to treatment with verapamil. Next, the sensitizing effects of verapamil on VIC-treated KBV20C cells were compared to those of pacritinib. As shown in Figure 4A and B, pacritinib (0.5 μM) and verapamil (10 μM) produced similar sensitization effects in cells treated with VIC, suggesting that lower pacritinib sensitizes VIC-treated KBV20C cells to levels similar to treatment with verapamil in sensitizing drug-resistant cancer cells over-expressing P-gp. Considering that pacritinib sensitizes KBV20C cells treated with VIC at low doses, it may be useful in clinical settings, given the minimal toxic concentrations in normal cells.
Jak2 inhibitors (pacritinib, CEP-33779, and NVP-BSK805) sensitize VIC-treated KBV20C cells via similar mechanisms of action. (A-B) KBV20C cells were plated on 60 mm-diameter dishes and treated with 5 nM vincristine, 0.5 μM pacritinib, 10 μM verapamil, 2 μM CEP-33779, 2 μM NVP-BSK805, 2 μM momelotinib, 2 μM WP1066, 5 nM VIC with 10 μM verapamil (VIC+VER), 5 nM VIC with 0.5 μM pacritinib (VIC+PACR), 5 nM VIC with 2 μM CEP-33779 (VIC+CEP-33779), 5 nM VIC with 2 μM NVP-BSK805 (VIC+NVP-BSK805), 5 nM VIC with 2 μM momelotinib (VIC+Momelotinib), 5 nM VIC with 2 μM WP1066 (VIC+WP1066), or 0.1% DMSO (Control). After 24 h, annexin V analyses (A) or microscopic observation at ×100 magnification (B) was performed as described in Materials and Methods.
Lower dose of pacritinib produced similar sensitization effects in VIC-treated cells with Jak2 inhibitors (fedratinib, CEP-33779, and NVP-BSK805) via similar mechanisms of action. Previously, Jak2 inhibitors demonstrated P-gp inhibitory activity and drug-sensitization effects in P-gp-over-expressing drug-resistant cancer cells. We compared the sensitization effects of pacritinib with those of Jak2 inhibitors (CEP-33779 and NVP-BSK805), which are known to sensitize drug-resistant cancer cells with P-gp inhibitory activity (19, 20). As seen in the degree of apoptosis induction in Figure 4A, 0.5 μM pacritinib, 2 μM CEP-33779, or 2 μM NVP-BSK805 showed similar efficacious sensitization in VIC-treated KBV20C cells. Therefore, considering the pacritinib, CEP-33779, and NVP-BSK805 results, we conclude that co-treatment of cells with Jak2 inhibitors can generally sensitize P-gp-over-expressing resistant cancer cells by inducing early apoptosis.
Furthermore, we compared the sensitization effects of the Jak2 inhibitor pacritinib with other Jak2 inhibitors (momelotinib and WP1066) (24). As seen in the degree of apoptosis induction and microscopic observations in Figure 4A and B, we found that both 2 μM momelotinib and 2 μM WP1066 showed much less sensitization effects in VIC-treated KBV20C cells, when compared to the co-treatment of 0.5 μM pacritinib, 2 μM CEP-33779, or 2 μM NVP-BSK805 with VIC. Therefore, considering the pacritinib, momelotinib, and WP1066 results, we conclude that not all Jak2 inhibitors in low doses can sensitize P-gp-over-expressing resistant cancer cells. This also suggests that the phase III Jak2 inhibitor pacritinib at low doses has a specific sensitization effect in P-gp-over-expressing resistant cancer cells.
Discussion
Drug repositioning is a popular practice for clinically used drugs (9, 11). It is advantageous as it leads to wider clinical applications in cancer patients, without additional toxicity tests. We have previously investigated many repurposed drugs, such as antimalarial drugs, anti-HIV drugs, anti-allergic drugs, antipsychotic drugs, and tyrosine kinase inhibitors, to determine their effects on drug-resistant cancer cells over-expressing P-gp (30-32, 34).
Jak2 inhibitors are usually administered to patients with types of blood cancers, such as leukemia, myelofibrosis, and myeloproliferative neoplasms (16, 17, 21-24). Among the commercially available Jak2 inhibitors, CEP-33779, NVP-BSK805, and XL019, lead to increased apoptosis of resistant cancer cells when administered as a combination therapy. These drugs can prevent the P-gp-mediated efflux of anticancer drugs (18-20). As they are not FDA-approved drugs, their clinical application may be delayed. Therefore, in this study, we evaluated the Jak2 inhibitor pacritinib that is currently in phase III trials and close to receiving FDA approval (21-24). We found that treatment with the pacritinib and VIC combination effectively sensitized drug-resistant KBV20C cancer cells. This will facilitate the quick application of pacritinib in patients, especially in those resistant to combination therapy with antimitotic drugs. Our findings can be applied to the co-treatment of other types of antimitotic drugs. We demonstrated that pacritinib exhibited similar sensitization effects on vinorelbine-, vinblastine-, and eribulin-treated resistant KBV20C cells. In particular, co-treatment with pacritinib and eribulin, a new antimitotic drug targeting MDR cancer cells (42, 43), had sensitization effects similar to those observed when administering the pacritinib–VIC combination. We hypothesized that pacritinib could be co-administered with various anticancer drugs to sensitize MDR cancer cells.
We investigated the molecular mechanisms underlying the co-treatment with VIC–pacritinib to accelerate the application of pacritinib in clinical settings, especially in patients resistant to antimitotic drugs. Using a more detailed quantitative annexin V study, we also demonstrated that VIC–pacritinib co-treatment significantly increased early apoptosis in KBV20C cells. Based on microscopy, FACS, and annexin V analyses, we concluded that early apoptosis was upregulated upon treatment with pacritinib, resulting from increased G2 arrest and reduced proliferation of drug-resistant KBV20C cells over-expressing P-gp. Furthermore, we found that the expression of the DNA damage-related marker pH2AX was elevated significantly, indicating high DNA damage, in addition to an increased number of cells undergoing early apoptotic death. Further in vivo studies using animal models will facilitate the quick application of pacritinib in patients with MDR. We found that pacritinib at a lower dose had P-gp-inhibitory activity, indicating that the increased apoptosis of VIC–pacritinib results from the inhibitory activity of pacritinib, preventing the efflux of VIC. Additionally, our measurements indicated that pacritinib showed efficacy similar to that of the established P-gp inhibitor verapamil at comparably lower doses. However, we found that pacritinib had less P-gp inhibitory activity than the positive controls, suggesting that it also removes or inhibits the factors that block antimitotic drugs in drug-resistant cancer cells and then exerts a synergistic effect in co-treated cells.
Further investigations of pacritinib are needed to determine the molecular targets that allow sensitization without P-gp inhibition. As P-gp inhibitors have shown toxicity towards normal cells (5), we believe that pacritinib should be considered as a co-treatment to sensitize drug-resistant cancer cells over-expressing P-gp. Since little increase in P-gp inhibition was detected for pacritinib when compared to verapamil and aripiprazole (positive controls), an improved combination of chemotherapeutic agents may be developed for cancer patients who develop resistance to antimitotic drugs. With the increasing popularity of personalized medicine, our results from the study of Jak2 inhibitors may contribute to making prescriptions for drug-resistant cancer patients more effective.
Various selective Jak2 inhibitors have been developed to improve Jak2 targeting (24, 44). The mechanism of action underlying the P-gp inhibitory functions of CEP-33779, NVP-BSK805, and XL019 has recently been demonstrated to sensitize MDR cancer cells (18-20). Therefore, the sensitization effects of pacritinib were compared with those of CEP-33779 and NVP-BSK805, as we also demonstrated that low doses of pacritinib have P-gp inhibitory activity. A lower dose of pacritinib showed similar efficacy to that of CEP-33779 or NVP-BSK805 in VIC-treated KBV20C cells, suggesting Jak2 inhibitors can sensitize VIC-treated resistant cancer cells by inducing early apoptosis. Notably, these Jak2 inhibitors when combined with VIC, exhibited similar sensitization mechanisms in P-gp-over-expressing resistant cancers. Furthermore, we investigated whether other Jak2 inhibitors (momelotinib and WP1066) at low doses had a sensitization effect on VIC-treated KBV20C cells. However, we did not observe similar sensitization effects with either momelotinib or WP1066, unlike those observed at low doses of pacritinib, CEP-33779, or NVP-BSK805. This suggests that not all Jak2 inhibitors sensitize MDR cancer cells. As pacritinib is close to being approved by the FDA, the urgent need for pharmacological treatments of antimitotic drug-resistant cancers can be efficiently addressed, and pacritinib may be used to treat P-gp-over-expressing resistant patients.
Acknowledgements
This work was supported by National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2019R1A2C2002923).
Footnotes
Authors’ Contributions
Yunmoon Oh and Jin-Sol Lee: Collected the data, contributed data or analysis tools, wrote the article. Ji Sun Lee and Jae Hyeon Park: Contributed data or analysis tools. Hyung Sik Kim and Sungpil Yoon: Contributed data or analysis tools, conceived and designed the analysis, collected the data, contributed data or analysis tools, wrote the article.
Conflicts of Interest
The Authors declare no conflicts of interest regarding this study.
- Received February 12, 2022.
- Revision received March 16, 2022.
- Accepted March 17, 2022.
- Copyright © 2022 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.