Abstract
Background/Aim: Co-treatment with calcineurin inhibitors, such as tacrolimus and cyclosporin A, can sensitize chemotherapy-resistant cancer cells with P-glycoprotein (P-gp)-over-expression. Pimecrolimus (PIME) is a clinically available calcineurin inhibitor with a structure similar to that of tacrolimus. Whether PIME can sensitize P-gp-over-expressing resistant cancer cells remains unclear. Materials and Methods: Cell viability assay, annexin V analyses, cellular morphology and density observation with a microscope, western-blotting, fluorescence-activated cell sorting (FACS), and analysis for P-gp inhibitory activity were performed to investigate the mechanism of action. Results: PIME exhibited strong cytotoxicity to vincristine (VIC)-treated drug-resistant cell lines (KBV20C and MCF-7/ADR) over-expressing P-gp. Co-treatment with VIC and PIME increased apoptosis and down-regulated the ERK signaling pathway, resulting in G2 arrest. PIME could be co-administered with vinorelbine or eribulin to sensitize resistant KBV20C or MCF-7/ADR cancer cells. Moreover, PIME strongly inhibited the efflux of both rhodamine 123 and calcein-AM substrates through P-gp after 4 h of treatment, indicating that VIC+PIME sensitized cancer cells by inhibiting VIC efflux via direct PIME binding to P-gp. Low doses of PIME, tacrolimus, and cyclosporin A showed similar sensitizing efficiencies in resistant KBV20C cells. These drugs showed similar P-gp inhibitory activities using both rhodamine 123 and calcein-AM substrates, suggesting that calcineurin inhibitors generally have strong P-gp inhibitory activities that sensitize drug-resistant cancer cells with P-gp over-expression. Conclusion: PIME, currently used in clinics, can be repositioned for treating patients with P-gp-over-expressing resistant cancer (stem) cells.
Vincristine (VIC), eribulin, vinorelbine, vinblastine, and paclitaxel (antimitotic drugs) target microtubules and prevent cellular division (1-5). Antimiotic or chemotherapeutic treatments can generally increase multidrug-resistant (MDR) cancer cells (6, 7). Therefore, it is important to identify novel agents or combination treatments for MDR cancer to improve the survival and prognosis of patients with MDR cancer.
MDR cancer cells can avoid the toxicity of antimitotic drugs through efflux by P-gp on the cellular membrane (6-9). P-gp over-expression in MDR cancer cells is the prominent mechanism to prevent cellular accumulation of toxic antimitotic drugs (8, 10-12). In addition, cancer cells can acquire resistant phenotypes through increased copy numbers of P-gp genes, which are over-expressed on the membranes following treatment with antimitotic drugs (8, 10-12). Thus, exploring mechanisms to sensitize P-gp-over-expressing MDR cancer cells could improve the treatment options for patients who develop resistance to antimitotic drugs. P-gp inhibitors have been developed for co-treatment with antimitotic drugs. However, their toxicity in normal cells and inhibition of cancer-targeting immune cells have prevented their use as treatment options (7, 12-15). Currently, novel P-gp inhibitors, with their reduced toxic or abnormal effects on normal cells while specifically targeting MDR cancer cells, are actively investigated (8-13).
We investigated novel combination treatments or P-gp inhibitors specifically targeting MDR cancer cells with less toxicity in normal cells. For this purpose, we focused on identifying US Food and Drug Administration (FDA)-approved drugs that could be repositioned. The toxicity of these agents is well known and can facilitate their rapid introduction to the treatment of patients with MDR cancer. Previously, we reported that drugs approved by FDA for indications other than cancer can highly sensitize P-gp-over-expressing MDR cancer cells (16-25). We assume that the identified drugs could be repositioned for the treatment of patients with MDR cancer as these drugs do not require additional toxicity testing (26-29).
The combination of calcineurin inhibitors, such as tacrolimus and cyclosporin A, can sensitize chemotherapeutic drug-treated MDR cancers through excellent P-gp inhibitory activity (14, 30-34). However, pimecrolimus (PIME), a calcineurin inhibitor recently approved by the FDA, has not been investigated in P-gp-over-expressing MDR cancer cells. In this study, we investigated whether co-treatment with PIME and VIC could sensitize P-gp-over-expressing resistant cancer cells. We also determined the cytotoxic mechanisms of this calcineurin inhibitor in combination with other antimitotic agents. Additionally, we compared the sensitization efficacies of PIME and other calcineurin inhibitors at low doses. Our results might facilitate the usefulness of PIME as a P-gp inhibitor in combination with therapeutic options for patients with P-gp-over-expressing MDR cancers.
Materials and Methods
Reagents. PIME, aripiprazole, tacrolimus, cyclosporin A, and tariquidar were purchased from Selleckchem (Houston, TX, USA). Verapamil and rhodamine 123 were purchased from Sigma-Aldrich (St. Louis, MO, USA). Calcein-AM was obtained from Invitrogen (Waltham, MA, USA). VIC and vinorelbine were obtained from Enzo Life Sciences (Farmingdale, NY, USA). An aqueous solution of eribulin (Eisai Korea, Seoul, Republic of Korea) was obtained from the National Cancer Center in South Korea.
C-PARP, pAkt, pGSK3β, pPDK1, pAMPK, p4EBP1, and pP70S6K antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). pErk1/2 and GAPDH antibodies were acquired from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
Cell lines. KBV20C and MCF-7/ADR, P-gp-over-expressing MDR cancer cell lines, were previously described in detail and used for a long period (16-25). To perform the experiments, the cells were grown in DMEM or RPMI1640 (WelGENE, Daegu, Republic of Korea) supplemented with fetal bovine serum (FBS) and antibiotics (penicillin and streptomycin).
Cell viability assay. Cellular viability was evaluated using an EZ-CyTox cell viability assay kit (Daeillab, Seoul, Republic of Korea). The assay has been described previously (24, 25, 35, 36). P-gp-over-expressing KBV20C cancer cells were grown in 96-well plates and treated with 2.5-5 nM VIC, 1.25-2.5 μM PIME, 1.25-2.5 μM aripiprazole, 1.25 μM tacrolimus, VIC+PIME, VIC+aripiprazole, VIC+tacrolimus, or 0.1% DMSO (negative control) for two days. Finally, 10 μl EZ-CyTox solution was added for 30-60 min at 37°C, and the absorbance at 450 nm was recorded using a VERSA MAX Microplate Reader (Molecular Devices Corp., Sunnyvale, CA, USA).
Quantitative analysis was performed from two independent experiments in triplicate. Data are presented as mean±standard deviation (SD). Statistical tests were performed using one-way analysis of variance (ANOVA) followed by Bonferroni’s test. We also confirmed our results using t-tests. Statistical significance was calculated using Graph Pad Prism Software Version 5.0 (GraphPad Software, San Diego, CA, USA). Statistical significance is indicated as follows: **p<0.01, ***p<0.001, and ****p<0.0001.
Annexin V analysis. Early and late apoptotic death was measured using a commercially available annexin V-fluorescein isothiocyanate (FITC) staining kit (BD Bioscience, Franklin, NJ, USA) according to previously described procedures (24, 25, 35, 36). P-gp-over-expressing KBV20C and MCF-7/ADR cancer cells were stimulated with 5-10 nM VIC, 1.25 μM PIME, 5 nM VIC+1.25 μM PIME, 10 nM VIC+1.25 μM PIME, or 0.1% DMSO (negative control) for one day. Pelleted cells were then stained with annexin V-FITC and propidium iodide and incubated for 30 min at 25°C. The stained cells were immediately examined using a Novocyte Flow cytometer (ACEA Biosciences, San Diego, CA, USA). The results were confirmed using at least two independent experiments.
Cellular morphology and density observation with a microscope. Cellular morphology and density were qualitatively examined using a microscope, as previously described (25, 35, 36). P-gp-over-expressing KBV20C or MCF-7/ADR cancer cells were cultured and stimulated with 5-10 nM VIC, 0.05 μg/ml vinorelbine, 30-60 nM eribulin, 1.25-5 μM PIME, 1.25 μM cyclosporin A, 1.25 μM tacrolimus, VIC+PIME, VIC+tacrolimus, VIC+cyclosporin A, vinorelbine+PIME, eribulin+PIME, or 0.1% DMSO (negative control) for 1-2 days. The treated cells were then observed using an ECLIPSETs2 inverted routine microscope (Nikon, Tokyo, Japan) with a ×40 or ×100 objective lens. The results were confirmed by at least two independent microscopic observations.
Western blotting. Total protein or phosphorylated protein levels were examined using western blot analysis, as previously described (22, 23, 25). P-gp-over-expressing KBV20C cancer cells were cultured and stimulated with 5 nM VIC, 1.25 μM PIME, 5 nM VIC+1.25 μM PIME, or 0.1% DMSO (negative control) for one day. Total cellular protein was isolated as described below. Pelleted cells were dissolved using the PRO-PREP™ kit (iNtRON, Seongnam, Republic of Korea) and incubated on ice for at least 20 min. Protein extracts from the supernatants were collected, and the protein concentrations were measured. Equal amounts of the protein extracts were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Western blot analysis was performed as previously described (22, 23, 25). The results were confirmed by at least two independent western blot experiments.
Fluorescence-activated cell sorting (FACS) analysis. Cell cycle analysis was performed as described previously (24, 25, 35, 36). P-gp-over-expressing KBV20C or MCF-7/ADR MDR cancer cells were stimulated with 5 nM VIC, 10 nM VIC, 1.25 μM PIME, 1.25 μM cyclosporin A, 1.25 μM tacrolimus, 5 nM VIC+1.25 μM PIME, 10 nM VIC+1.25 μM PIME, 5 nM VIC+1.25 μM cyclosporin A, 5 nM VIC+1.25 μM tacrolimus, or 0.1% DMSO (negative control) for one day. Pelleted cells were then dissolved in 75% ethanol for 1 h at −20°C and stained with propidium iodide for 0.5 h. The treated cells were observed using a Novocyte Flow Cytometer (ACEA Biosciences). The results were confirmed by at least two independent FACS experiments.
Analysis for P-gp inhibitory activity. Uptake assays for rhodamine 123 and calcein-AM were performed as previously described to quantitatively measure the inhibitory activity of calcineurin inhibitors on P-gp (20, 22, 23). KBV20C or MCF7/ADR cell lines were stimulated with low doses of positive controls (aripiprazole, tariquidar, or verapamil) or calcineurin inhibitors (PIME, tacrolimus, or cyclosporin A). After treatment for 1 h at 37°C, fresh medium was changed, and cells were stained with either rhodamine 123 (0.5 μM) or calcein-AM (0.1 μg/ml) for 3 h. Stained pelleted cells were quantitatively examined using a Novocyte Flow cytometer (ACEA Biosciences). The results were confirmed by at least two independent FACS experiments.
Results
PIME co-treatment increases the cytotoxicity of VIC to the MDR cancer cell line, KBV20C. Calcineurin inhibitors, such as tacrolimus and cyclosporin A, sensitize MDR cancer cells (14, 30, 32, 37). Therefore, identifying novel calcineurin inhibitors and their mechanisms of action in drug-resistant cancers is an important avenue for broadening their clinical application.
In this study, we evaluated PIME, a calcineurin inhibitor recently approved by the FDA (37, 38). We used P-gp-over-expressing MDR KBV20C cancer cells with an antimitotic drug-resistant phenotype (22, 23, 25). As shown in Figure 1A, treatment with PIME alone had no effect on KBV20C cells. However, co-treatment with 2.5 or 5 μM PIME significantly reduced the viability of VIC-treated KBV20C cells. We previously demonstrated that aripiprazole could increase the cytotoxicity of VIC to MDR cancer cells (17, 20, 23). Similar to aripiprazole (positive control), PIME increased the cytotoxicity of VIC in KBV20C cancer cells (Figure 1B). Collectively, our results indicate that PIME can significantly increase the cytotoxic effects of antimitotic drugs in MDR cancer cells.
Co-treatment with PIME increases the cytotoxicity of VIC to P-gp-over-expressing drug-resistant KBV20C cancer cells. (A) Drug-resistant KBV20C cells were grown on 60 mm-diameter dishes and treated with 5 nM VIC, 2.5 μM PIME, 5 μM PIME, 5 nM VIC with 2.5 μM PIME (VIC+PIME-2.5), 5 nM VIC with 5 μM PIME (VIC+PIME-5), or 0.1% DMSO (CON). After one day, all cells were observed using an inverted microscope at ×40 magnification. (B) KBV20C cells were seeded in 96-well plates and grown to 30%-40% confluence. These cells were then stimulated for 48 h with 5 nM VIC, 2.5 μM PIME, 2.5 μM aripiprazole (ARI), 5 nM VIC with 2.5 μM PIME (PIME+VIC), 5 nM VIC with 2.5 μM aripiprazole (ARI+VIC), or 0.1% DMSO (CON). Cell viability assay was performed as described in Materials and Methods. Data are presented as mean±standard deviation (SD) of at least two experiments repeated in triplicate. ****p<0.0001 was considered statistically significant. (C-D) KBV20C cells were grown on 60 mm-diameter dishes and treated with 5 nM VIC, 1.25 μM PIME, 5 nM VIC with 1.25 μM PIME (VIC+PIME), or 0.1% DMSO (CON). After 24 h, annexin V (C) or FACS analyses (D) were performed as described in Materials and Methods. (E) KBV20C cells were seeded in 60 mm-diameter dishes and treated with 5 nM VIC, 1.25 μM PIME, 5 nM VIC with 1.25 μM PIME (VIC+PIME), or 0.1% DMSO (CON). After 24 h, western blot analysis was performed using antibodies against C-PARP, pAMPK, pGSK3β, pPDK1, pErk1/2, p4EBP1, pP70S6K, pAkt, and GAPDH.
Co-treatment with VIC and PIME induces apoptosis of KBV20C cells. Cell apoptosis was evaluated using annexin V staining. When the proportion of early and late apoptotic cells was quantitatively estimated, VIC+PIME co-treatment significantly increased apoptotic cells compared with VIC treatment alone (Figure 1C). This suggests that the combination of VIC and PIME can induce early apoptosis and increase late apoptosis, thereby causing rapid cellular death. We then measured the expression levels of the apoptotic marker C-PARP to confirm apoptotic cell death following VIC+PIME co-treatment (22, 23, 25). As shown in Figure 1E, cells co-treated with VIC+PIME showed increased C-PARP expression, demonstrating that co-treatment with PIME can sensitize VIC-treated KBV20C-resistant cancer cells via an apoptotic mechanism.
VIC+PIME co-treatment increases the proportion of resistant KBV20C cells in the G2 phase. We performed FACS analyses to measure cell cycle arrest following VIC+PIME co-treatment. As shown in Figure 1D, VIC+PIME co-treatment caused G2 arrest in KBV20C cells when compared with cells treated with VIC alone. Western blot analysis was performed to determine the expression levels of G2 arrest-related proteins (39, 40). Notably, pErk levels were largely reduced following VIC+PIME co-treatment (Figure 1E), suggesting that the ERK pathway might increase the number of VIC+PIME-co-treated resistant KBV20C cancer cells in the G2 phase.
Low doses of PIME strongly and rapidly inhibit P-gp activity in both rhodamine 123 and calcein-AM substrate efflux assays. To investigate the mechanisms involved in the effect of PIME co-treatment, we examined P-gp inhibition by PIME in resistant KBV20C cancer cells. We hypothesized that co-treatment with PIME could sensitize VIC-treated KBV20C cells via P-gp inhibition.
Aripiprazole, tariquidar, and verapamil strongly inhibit P-gp activities (20, 22, 23, 25). Thus, they were used as positive controls for comparison with PIME. We tested the extent to which KBV20C cells accumulated rhodamine 123 or calcein-AM (two P-gp substrates) following treatment with a potential P-gp inhibitor or positive control (22, 23).
As shown in Figure 2A, P-gp inhibitory activity was approximately 2.5- to 4-fold higher in aripiprazole, tariquidar, and verapamil-treated cells than in untreated controls. Similar to positive controls, PIME inhibited P-gp (Figure 2A) and reduced the efflux of both rhodamine 123 and calcein-AM substrate. This suggests that P-gp inhibition by PIME contributes to the sensitizing effect of the VIC+PIME co-treatment. PIME showed strong P-gp inhibition after only a short treatment time (4 h) (Figure 2A). This indicates that PIME plays a role in inhibiting P-gp activity by directly binding to P-gp and not by regulating P-gp transcription or translation.
Low doses of PIME exert strong P-gp inhibitory activity after 4 h treatment in both rhodamine 123 and calcein-AM substrate efflux assays. (A) P-gp-over-expressing drug-resistant KBV20C cells were treated with 2.5 μM aripiprazole (ARI), 5 μM PIME, 2.5 μM PIME, 1.5 μM tariquidar, 10 μM verapamil (VER), or 0.1% DMSO (CON). After 1 h, all cells were stained with rhodamine 123 (upper) or calcein-AM (lower) for 3 h and used for FACS analysis as described in Materials and Methods. (B) Drug-resistant KBV20C cells were grown in 60 mm-diameter dishes and treated with 2.5 μM PIME, 60 nM eribulin, 0.05 μg/ml vinorelbine (VIO), 0.05 μg/ml vinorelbine with 2.5 μM PIME (PIME+vinorelbine), 60 nM eribulin with 2.5 μM PIME (PIME+eribulin), or 0.1% DMSO (CON). After one day, all cells were observed using an inverted microscope at ×100 magnification. (C) KBV20C cells were seeded in 96-well plates and grown to 30%-40% confluence. These cells were then stimulated for 48 h with 2.5 nM VIC, 1.25 μM tacrolimus (TARC), 1.25 μM PIME, 1.25 μM aripiprazole (ARI), 2.5 nM VIC with 1.25 μM tacrolimus (TARC+VIC), 2.5 nM VIC with 1.25 μM PIME (PIME+VIC), 2.5 nM VIC with 1.25 μM aripiprazole (ARI+VIC), or 0.1% DMSO (CON). Cell viability assay was performed as described in Materials and Methods. Data are presented as mean±standard deviation (SD) of at least two experiments repeated in triplicate. ***p<0.001; ****p<0.0001.
Co-treatment with PIME sensitizes vinorelbine or eribulin-treated KBV20C cells. Next, we examined whether PIME could also sensitize other antimitotic drug-treated KBV20C cells to determine the general applicability of PIME in combination treatments. We tested vinorelbine and eribulin, two antimitotic drugs that are routinely used (2, 23). As shown in Figure 2B, vinorelbine+PIME and eribulin+PIME treatments showed increased cytotoxicity against resistant KBV20C cells, as observed with VIC+PIME co-treatment. These results suggest that PIME could be a combination partner of various antimitotic drugs for sensitizing drug-resistant cancer cells over-expressing P-gp.
Low doses of PIME and tacrolimus exhibit similar sensitizing effects. Furthermore, we investigated whether co-treatment with other calcineurin inhibitors could sensitize drug-resistant cancer cells (37, 38). As shown in Figure 2C, low doses of PIME and tacrolimus exhibited cytotoxic effects similar to those observed with aripiprazole (the positive control) in a viability assay when combined with VIC in KBV20C cells. As tacrolimus shows strong P-gp inhibition (14, 30), we can conclude that co-treatment with calcineurin inhibitors and antimitotic drugs can sensitize antimitotic drug-treated cancer cells.
Low doses of PIME and cyclosporin A, another calcineurin inhibitor, exhibit similar cytotoxic effects. There are two types of structurally distinct calcineurin inhibitors (14, 37, 38). Tacrolimus and PIME have similar chemical structures, whereas cyclosporin A has a different structure and ring-forming backbone. Although co-treatment with tacrolimus or cyclosporin A strongly inhibits P-gp, resulting in sensitization of drug-resistant cancer cells (14, 30, 31, 33), a comparison of low-dose PIME, tacrolimus, and cyclosporin A has not yet been reported. As shown in Figure 3A, low doses of the three calcineurin inhibitors exhibited strong cytotoxic effects on KBV20C cells. We also confirmed that all three calcineurin inhibitors induced G2 arrest to a similar extent in VIC-treated KBV20C cells (Figure 3B). We concluded that calcineurin inhibitors can sensitize antimitotic drug-resistant cancer cells via strong P-gp inhibition.
Low doses of PIME and other types of calcineurin inhibitors (tacrolimus and cyclosporin A) exhibit similar cytotoxic effects on P-gp-over-expressing KBV20C cancer cells. (A-B) KBV20C cells were grown in 60 mm-diameter dishes and treated with 5 nM VIC, 1.25 μM PIME, 1.25 μM tacrolimus (TARC), 1.25 μM cyclosporin A (CYCL), 5 nM VIC with 1.25 μM PIME (VIC+PIME), 5 nM VIC with 1.25 μM tacrolimus (VIC+TARC), 5 nM VIC with 1.25 μM cyclosporin A (VIC+CYCL), or 0.1% DMSO (CON). After 24 h, microscopic (A) and FACS analyses (B) were performed as described in Materials and Methods. (C) P-gp-over-expressing drug-resistant KBV20C cells were treated with 2.5 μM aripiprazole (ARI), 2.5 μM PIME, 2.5 μM tacrolimus (TARC), 2.5 μM cyclosporin A (CYCL), 10 μM verapamil (VER), or 0.1% DMSO (CON). After 1 h, all cells were stained with rhodamine 123 or calcein-AM for 3 h and used for FACS analysis as described in Materials and Methods.
PIME, tacrolimus, and cyclosporin A exert similar strong P-gp inhibition. Furthermore, we compared the degree of P-gp inhibition by calcineurin inhibitors (PIME, tacrolimus, and cyclosporin A) at the same dose. We measured the substrate specificity of P-gp inhibition using two substrates, rhodamine 123 and calcein-AM. We hypothesized that the similar P-gp inhibitory activities of rhodamine 123 and calcein-AM substrates reflected functional inhibition of the same P-gp domain in the membrane.
As shown in Figure 3C, aripiprazole and verapamil-treated cells exhibited 2.5- and 5-fold higher P-gp inhibition than DMSO-treated controls. All the three calcineurin inhibitors studied (PIME, tacrolimus, and cyclosporin A) inhibited P-gp at the same dose in a manner similar to aripiprazole or verapamil (Figure 3C), as shown in both the rhodamine 123 and calcein-AM substrate efflux assays. This suggests that co-treatment with calcineurin inhibitors contributed to the inhibition of P-gp and subsequently of VIC efflux in VIC-treated resistant KBV20C cells. These findings indicate that calcineurin inhibitors generally exhibit similar substrate specificity and P-gp-inhibitory mechanisms to prevent the efflux of antimitotic drugs.
In addition to well-known P-gp inhibitory drugs, such as tacrolimus and cyclosporin A, low-dose PIME might also be useful in the clinic for treating specific types of P-gp-related resistant cancer because PIME, tacrolimus, and cyclosporin A can selectively target P-gp-over-expressing drug-resistant cancers with detailed cancer-specificity.
VIC+PIME also sensitizes P-gp-over-expressing MCF-7/ADR cancer cells. We also investigated whether VIC+PIME could be employed for other types of P-gp-over-expressing resistant cancers. MCF-7/ADR cancer cells are routinely used to study drug sensitization for drug-resistant cancers (23). Thus, we used MCF-7/ADR drug-resistant breast cancer cells in the present study. MCF-7/ADR cells exhibit a higher P-gp expression than KBV20C cells (23, 41). As shown in Figure 4A, PIME sensitized VIC-treated MCF-7/ADR cancer cells. As a positive control, aripiprazole was administered at the same dose as PIME. We observed that PIME and aripiprazole showed similar sensitization effects in VIC-co-treated MCF-7/ADR cells. As shown in Figure 4B, eribulin+PIME markedly reduced the growth of MCF-7/ADR cells, suggesting that PIME can be administered in combination with other chemotherapeutic drugs to sensitize resistant cancer cells. Both annexin V and FACS analyses were performed to quantitatively evaluate the cytotoxicity of VIC+PIME to MCF-7/ADR cells. As shown in Figure 4C and D, VIC+PIME increased apoptosis and G2 arrest. This finding suggests that PIME increases the cytotoxicity of VIC in multiple types of P-gp-over-expressing drug-resistant cancer cells. Furthermore, we confirmed that PIME significantly inhibited P-gp in MCF-7/ADR cells in a manner similar to that of the positive controls, as shown in both rhodamine 123 and calcein-AM substrate efflux assays. Taken together, these results indicate that low-doses of PIME can sensitize antimitotic drug-resistant KBV20C or MCF-7/ADR cells via strong inhibitory activity in the substrate efflux of P-gp.
Co-treatment with PIME increases the cytotoxicity of VIC to P-gp-over-expressing resistant MCF-7/ADR cancer cells. (A) Drug-resistant MCF-7/ADR cells were grown in 60 mm-diameter dishes and treated with 10 nM VIC, 10 nM VIC with 2.5 μM PIME (VIC+PIME), 10 nM VIC with 2.5 μM aripiprazole (VIC+ARI), or 0.1% DMSO (CON). After two days, cells were observed using an inverted microscope at ×100 magnification. (B) MCF-7/ADR cells were grown in 60 mm-diameter dishes and treated with 2.5 μM PIME, 30 nM eribulin, 30 nM eribulin with 2.5 μM PIME (PIME+ERI), or 0.1% DMSO (CON). After one day, cells were observed using an inverted microscope at ×100 magnification. (C-D) MCF-7/ADR cells were grown in 60 mm-diameter dishes and treated with 10 nM VIC, 1.25 μM PIME, 10 nM VIC with 1.25 μM PIME (VIC+PIME), or 0.1% DMSO (CON). After 24 h, annexin V (C) and FACS analyses (D) were performed as described in Materials and Methods. (E) P-gp-over-expressing drug-resistant MCF-7/ADR cells were treated with 5 μM PIME, 2.5 μM PIME, 1.5 μM tariquidar (Tari), 10 μM verapamil (VER), or 0.1% DMSO (CON). After 1 h, cells were stained with rhodamine 123 or calcein-AM for 3 h and used for FACS analysis as described in Materials and Methods.
Discussion
Drug repositioning is a popular practice for identifying new clinical drugs for cancer treatments (26-29). The time interval for toxicity testing can be reduced once novel drug repositioning or its mechanisms have been elucidated. Thus, drug repositioning can be easily used in patients with resistant cancers. We have previously identified various repositioned drugs for P-gp-over-expressing drug-resistant cancer cells (18-24, 42-46). These drugs include specific molecule-targeting inhibitors, such as inhibitors of JAK2, tyrosine or serine kinase, virus proteases, and histamine or dopamine receptors.
Calcineurin inhibitors can reverse resistance to cancer treatments. They have strong P-gp inhibitory activity (14, 30-34, 37, 38). Tacrolimus and cyclosporin A showed enhanced cytotoxicity in P-gp-over-expressing drug-resistant cancer cells when administered in combination. These drugs have P-gp-inhibitory activity and can prevent the P-gp-mediated efflux of chemotherapeutic drugs (14, 30, 31). Calcineurin inhibitors can be repositioned to treat patients with resistant cancer. Therefore, in this study, we evaluated whether PIME, a calcineurin inhibitor that has recently received FDA approval (37, 38), could sensitize P-gp-over-expressing drug-resistant cancer cells.
Co-treatment with PIME significantly sensitized drug-resistant KBV20C and MCF-7/ADR cancer cells to VIC. For the first time, we found that PIME is highly cytotoxic to P-gp-over-expressing drug-resistant cancer cells. Our results will facilitate the rapid application of PIME in combination therapy for patients with drug-resistant cancer. We also confirmed the strong sensitization effects of PIME on vinorelbine- or eribulin-treated resistant KBV20C and MCF-7/ADR cells. In particular, co-treatment with PIME and eribulin, a promising drug recently developed for the treatment of resistant cancers (47, 48), showed sensitization effects similar to those observed with VIC+PIME in resistant cancer cells. We hypothesized that PIME could be used in combination with various antimitotic drugs to sensitize drug-resistant cancer cells.
The molecular mechanisms for VIC+PIME sensitization have been analyzed to accelerate its application in a clinical setting. We demonstrated that co-treatment with PIME reduced viability and increased the number of both KBV20C and MCF-7/ADR resistant cancer cells in the G2 phase. We also demonstrated that VIC+PIME co-treatment increased early apoptosis in resistant KBV20C cells. Based on these analyses, we conclude that PIME induces early apoptosis, resulting from increased G2 arrest. Furthermore, an investigation of the expression levels of proteins involved in cellular signaling pathways in KBV20C cells co-treated with VIC and PIME revealed that the levels of pErk were significantly reduced. This indicated a mechanism involving G2 phase arrest via the ERK pathway. To facilitate the rapid application of PIME combination therapy in patients, we should consider further in vivo xenograft studies using animal models.
We also evaluated whether the sensitization effect of VIC+PIME was a result of P-gp inhibition by PIME. Low-dose PIME had a strong P-gp-inhibitory activity, preventing the efflux of VIC and resulting in the sensitization of cancer cells to VIC+PIME. PIME showed efficacy similar to that of established strong P-gp inhibitors, such as aripiprazole, verapamil, and tariquidar (positive controls), at low doses. We further demonstrated that a short-term (4 h) treatment with PIME had a strong P-gp inhibitory activity, suggesting that PIME could inhibit P-gp by direct binding. PIME had strong P-gp inhibitory activity against the efflux of two different substrates, rhodamine 123 and calcein-AM. Additionally, we confirmed the mechanisms involved in the strong P-gp-inhibitory activity of PIME in two P-gp-over-expressing drug-resistant cancer cell lines, KBV20C and MCF-7/ADR. Therefore, PIME can be a substitute for other P-gp inhibitors in drug repositioning, replacing P-gp inhibitors that are toxic to normal cells. Our results suggest that PIME could be considered as a co-treatment to specifically target P-gp-over-expressing drug-resistant cancer cells owing to its strong P-gp inhibitory activity. As personalized medicine is gaining popularity, our findings on PIME might contribute to effective treatment options for patients with P-gp-over-expressing drug-resistant cancer.
Furthermore, we investigated whether low doses of certain calcineurin inhibitors (PIME, tacrolimus, and cyclosporin A) have a similar sensitizing effect on VIC-treated KBV20C cells. Low doses of PIME showed efficacy similar to that of tacrolimus or cyclosporin A in sensitizing VIC-treated KBV20C cells, suggesting that the co-treatment of cells with VIC and any calcineurin inhibitor could sensitize P-gp-over-expressing drug-resistant cancer cells by inducing G2 arrest. In particular, we observed similar sensitization effects with PIME, tacrolimus, and cyclosporin A, indicating that the strong P-gp inhibitory activity of calcineurin inhibitors significantly contributes to the increased cytotoxic effects of antimitotic drugs in P-gp-over-expressing drug-resistant KBV20C cancer cells. Calcineurin inhibitors with different structures can be used clinically as alternative backup inhibitors for drug-resistant cancer cells. We hypothesized that in a clinical setting, combination therapy with these calcineurin inhibitors could be applied to increase the drug sensitivity of P-gp-over-expressing cancer cells in heterogeneous tumor cell populations.
As personalized medicine is gaining popularity, our results from this study might contribute to the goal of making prescriptions for patients with drug-resistant cancer effective. Although the resistant cancer-sensitizing abilities of the calcineurin inhibitors have been previously demonstrated (14, 30-32), our findings support the application of PIME and other examined calcineurin inhibitors as potential drug-repurposing candidates.
Comparing the P-gp inhibition mediated by aripiprazole, verapamil, and calcineurin inhibitors at the cellular level, PIME can be expected to induce P-gp inhibition at nanomolar concentrations in an in vitro test tube assay. In future work, in vitro assays should be employed to determine whether these calcineurin inhibitors can directly bind to P-gp to inhibit its activity. Considering that the toxicity of PIME is well documented in clinics, it can be efficiently applied to the urgent need of treatment for patients with antimitotic drug-resistant cancers.
Acknowledgements
The Authors thank YUNMOON OH for technical support and the preparation of the manuscript. This work was supported by the National Research Foundation of Korea (NRF), funded by the Ministry of Education (NRF-2019R1A2C2002923).
Footnotes
Authors’ Contributions
Jae Hyeon Park, Ji Sun Lee, Joo Kyung Shin, and Swati Sharma: Collected the data, contributed data and analysis tools, and wrote the article. Jae Hyeon Park, Ji Sun Lee, Joo Kyung Shin, Hyung Sik Kim: Contributed data and analysis tools. Sungpil Yoon: Contributed the data and analysis tools, conceived, and designed the analysis, collected the data, contributed data or analysis tools, and wrote the article.
Conflicts of Interest
The Authors declare no conflicts of interest regarding this study.
- Received December 28, 2022.
- Revision received January 26, 2023.
- Accepted January 27, 2023.
- Copyright © 2023 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.
