Our previous study has shown that perphenazine and prochlorperazine induce a concentration-dependent loss in human glioblastoma U87-MG viability; additionally, the obtained EC50 values for the drugs (0.98 and 0.97 µM, respectively) (Otręba and Buszman 2018) correlate with the concentrations detected in human plasma. It was shown that perphenazine and prochlorperazine in the concentration of 0.5 and 1.0 µM reduced U87-MG cells viability by 32 and 54.5 as well as 30.5 and 56.3 %, respectively (Otręba and Buszman 2018). In the present study, we tested the impact of perphenazine and prochlorperazine in the U87-MG cell line on a level of ABC transporters, trying to explain the differences observed in the cellular viability. Obtained results demonstrating that the analyzed phenothiazine derivatives can reduce ABCB1 and increase ABCG2 content in glioblastoma cells. Since ABCB1 also regulates cell proliferation, and the knockdown of ABCB1 suppresses cell proliferation (Muriithi et al. 2020), it can explain the decrease of cellular viability after perphenazine and prochlorperazine treatment because in our study we observed a decrease of ABCB1 amount. It was found also that prochlorperazine significantly reduces the rate of cell migration.
ABCB1 and ABCG2 transporters are localized in the brain endothelial capillaries (Balça-Silva et al. 2018) and are related to multidrug resistance (Liu et al. 2016). In certain glioblastomas, high expression of ABCB1 and ABCG2 has been reported to associate with poor prognosis. It is believed that the transporters that limit drug accumulation are upregulated in the tumor cells thereby limiting drug uptake (Liu et al. 2016). Interestingly, the resistance to TMZ, which is the drug of choice in treating glioblastoma, may be related to the excretion of the drug from the brain by these transporters (Wijaya et al. 2017). It was confirmed by a study, which showed the increase TMZ accumulation in the tumors of mouse GBM model during elacridar treatment (non-selective inhibitor of ABCB1 (Chen et al. 2017) as well as in mice that were deficient for ABCB1 and ABCG2 genes (Lin et al. 2014). The ABCG2 transporter protects tissues against deadly xenobiotic exposures by the contribution of the absorption, distribution, and elimination of the drugs and endogenous compounds (Gupta et al. 2020). Moreover, the ABCG2 expression enhances stress-induced autophagy and cell survival in multiple tumor cell types (Wesołowska 2011).
Antipsychotic phenothiazines such as chlorpromazine (Abdallah et al. 2015, Wesołowska 2011), prochlorperazine (Wesołowska 2011), thioridazine, and fluphenazine (Abdallah et al. 2015) impair drug efflux mediated by ABCB1 demonstrating an inhibitor activity. Noteworthy, verapamil and thioridazine not only block ABCG2 or ABCB1 (P-gp) but also can reverse the resistance of doxorubicin-resistant sarcoma 180 cells completely. Spengler et al. (2016) suggest that thioridazine, in concentrations greater than that clinically employed for the therapy of severe psychosis, induces apoptosis of the MDR lymphoma cells and inhibits the activity of their over-expressed ABCB1 transporter. Wang et al. (2008) showed that antipsychotic drugs (e.g. chlorpromazine) exhibited moderate to mild inhibitory effects on BCRP (ABCG2) activity and its transport efficiency in breast cancer MCF7 and MCF7/MX100 cells. The authors based on their previous findings suggest that several antipsychotics (chlorpromazine, risperidone, quetiapine, paliperidone, and clozapine) are dual inhibitors of both P-gp and BCRP. Interestingly, Riganti et al. (2014) demonstrated that TMZ dose-dependent decreases ABCB1 level, without affecting ABCG2 in the immortalized primary human brain microvascular endothelial cell line (hCMEC/D3). It confirms our findings in the ABCB1 amount observed in the concentration of 0.1 µM of perphenazine and prochlorperazine. Moreover, in case of both drugs significant increase of the P-gp amount was dose-dependent. Interestingly the observed decrease of ABCB1 amount was not caused by cell death or proliferation disturbances, since previous results of the WST-1 assay showed that perphenazine in the concentration of 0.1 µM does not affect U-87MG cells viability (Otręba and Buszman 2018).
Pan et al. (2017) measured protein level and gene expression of ABCG2 in four different human malignant glioma cell lines (A172, U87, SHG-44, and U251). Western blot analysis showed that U87 has the lowest ABCG2 amount among all the cell lines, whereas no significant differences are found in the mRNA expression levels of MRP1 and MDR1 in the four GBM cells.
We also evaluated the effect of perphenazine and prochlorperazine on wound closure, transwell migration, and transwell migration of human glioblastoma cell line since migrating cells at the marginal zones of GMB tumors are less sensitive to apoptosis leading in consequence to the frequent recurrences (Sarafian et al. 2009). The wound-healing assay showed a time-dependent increase in wound area closure. The significant differences were observed between the time t0 and 3 hours for control and perphenazine (p < 0.01) as well as between t0 and 6, 9, 12 as well as 24 hours for control, perphenazine, and prochlorperazine (p < 0.01). Moreover, stronger stimulation of U87-MG migration after perphenazine (1.0 µM) treatment was observed after 6-hour treatment in comparison to prochlorperazine. The analysis of the rate of cell migration after 24 h incubation showed the tendency that the U-87MG cells in perphenazine or prochlorperazine migrate slower in comparison to control. Interestingly, only in case of prochlorperazine, the difference is statistically significant (p < 0.01) and suggests that the cells migrate 1.3 times slower in prochlorperazine (1,0 µM) in comparison to control.
Moreover, we analyzed the transwell migration of U87-MG cells using the drug concentration causing only about a 30% decrease in cell viability. The study showed that both of the analyzed drugs can decrease migration and invasion of the cells. Noteworthy, the analysis of internal control shown that perphenazine (0.5 µM), and prochlorperazine (0.5 µM) may be a chemoattractant for cellular invasion and migration but 5–6 times weaker and 3–4 times weaker, respectively than the growth medium with 10% FBS. It may also explain that less percentage of the cells invaded and/or migrated from the growth medium with 1% FBS and perphenazine or prochlorperazine to the growth medium with 10% FBS in comparison to cell migration/invasion from the growth medium with 1% FBS to the growth medium with 10% FBS.
The observed strong effect of perphenazine and prochlorperazine on viability and migration of human glioblastoma may be related to ABCB1 and/or ABCG2 amounts, since the proteins may affect cell migration. P-Glycoprotein contains a Pim-1 phosphorylation consensus sequence, which mediates the phosphorylation of Ser683 in P-glycoprotein, protects ABCB1 from degradation, and allows its glycosylation and cell-surface expression. Overexpression of the serine/threonine protein kinase Pim-1 is often observed in human malignancy tumors (acute myeloid leukemia, acute lymphoblastic leukemia, prostate cancer, and gastric cancer (Katayama et al. 2014), breast cancer (Brasó-Maristany et al. 2016), glioblastoma multiforme (Herzog et al. 2015). Noteworthy, Pim-1 promotes tumor cell growth by promoting cell cycle progression, cell migration, and protein translation and by the suppression of apoptosis (Brasó-Maristany et al. 2016). Moreover, Liang et al. (2015) showed that nuclear ABCG2 in lung cancer is involved in a transcription regulation of the E-cadherin–encoding gene (CDH1). CDH1 is a key cell-cell adhesion gene. Thus, ABCG2 could affect cell migration, since the ABCG2 overexpression enhances E-cadherin expression as well as increased nuclear ABCG2 expression (Liang et al. 2015). Especially if E-cadherin prevents loss of cell-cell adhesion and cell junctions, which allow cells to invasion and migration (Na et al. 2020). Nucleus localization of ABCG2 was also confirmed in glioblastoma multiforme (Bhatia et al. 2012) as well as relative expression of E-cadherin by Western blot was shown in U87-MG cells (Zhang et al. 2015). Thus, ABCG2 may also affect migration in U87-MG. Our studies have shown a significant increase of ABCG2 after prochlorperazine treatment (0.1 and 1.0 µM) of U-87 MG cells as well as E-cadherin after perphenazine (0.25 and 0.5 µM) and prochlorperazine treatment (0.25 µM) what is in line with the above suggestions.
Interestingly, the alteration of alpha-tubulin acetylation “may be involved in cancer cell migration, invasion, and cancer metastasis” (Lee et al. 2015). It is possible since the acetylation may promote cellular migration by the increase in the vicinity of focal adhesions (Bance et al. 2019).
The α3 integrin is mainly localized in invading cells and cells surrounding vessels of glioblastoma. Moreover, the overexpression of α3 leads to the increase of glioma migration and/or invasion, while “downregulation of α3 integrin inhibits glioma invasion concomitant with a change in the phosphorylation level of the extracellular signal-regulated kinase (ERK) 1/2 pathway”. Thus, α3 integrin plays important role in the cellular invasion by ERK 1/2 activation (Nakada et al. 2013). In case of α5 integrin, it plays important role in cellular migration, invasion, and adhesion of many tumors including glioblastoma. The α5β1 integrin is also called the critical regulator of cell migration and invasion since it can affect cytoskeleton rearrangement, cell adhesion, and the production of matrix metalloproteinase (MMP). The integrin may promote cancer cell invasion and metastasis by the activation of the c-Met/FAK/ Src-dependent signaling pathway or regulation of the expression and activity of MMPs (Hou et al. 2020). Nakada et al. 2013 showed that overexpression of the α3 integrin in glioblastoma cells (U87-MG, SNB19, and U251) increases cell migration. Noteworthy, in U87-MG cells the authors observed the highest expression of α3 integrin (1.3 fold) and the lowest invasion of glioblastoma cells (1.5 fold). The analysis of invasion showed that the invasion of U87-MG cells was stronger in α3 integrin overexpressing cells, which suggests that α3 integrin may be an invasion promotor. Mallawaartchy et al. 2015 showed a high level of α5 integrin in U87-MG cells. The authors also identified 49 proteins connected with cell invasion. Moreover, the gene expression data of α5 integrin showed “prognostic significance in independent glioblastoma cohorts”. Those observations are in line with our results since we observed a decrease of migration and invasiveness of U87-MG cells after treatment with perphenazine or prochlorperazine on the concentration 0.5 µM. In the same concentration, we did not notice the increased level of, which may explain a decrease in cellular migration and invasion.
Furthermore, α5β1 integrin also precipitates in the aggressiveness of solid tumors, the high expression of the protein may decrease patient survival, which makes it an important factor in therapy resistance (Renner et al. 2016). Thus, the noticed in our study trend to decrease of α5 and β1 integrins may be a good prognosis in glioblastoma therapy.
Finally, Li et al (2016) showed that bacitracin can be used as an anti-invasive agent, which is in line with our results.
In the future, we plan to use PCR assay to confirm variations of the proteins as well as use more human glioblastoma cell lines to get more generalized conclusions about the possibility of use phenothiazine derivatives in glioblastoma treatment.