Pimavanserin: A Novel Autophagy Modulator for Pancreatic Cancer Treatment

Abstract

Pancreatic tumors exhibit high basal autophagy compared to that of other cancers. Several studies including those from our laboratory reported that enhanced autophagy leads to apoptosis in cancer cells. In this study, we evaluated the autophagy and apoptosis inducing effects of Pimavanserin tartrate (PVT). Autophagic effects of PVT were determined by Acridine Orange assay and Transmission Electron Microscopy analysis. Clinical significance of ULK1 in normal and pancreatic cancer patients was evaluated by R2 and GEPIA cancer genomic databases. Modulation of proteins in autophagy signaling was assessed by Western blotting and Immunofluorescence. Apoptotic effects of PVT was evaluated by Annexin-V/APC assay. Subcutaneous xenograft pancreatic tumor model was used to evaluate the autophagy-mediated apoptotic effects of PVT in vivo. Autophagy was induced upon PVT treatment in pancreatic ducal adenocarcinoma (PDAC) cells. Pancreatic cancer patients exhibit reduced levels of autophagy initiator gene, ULK1, which correlated with reduced patient survival. Interestingly, PVT induced the expression of autophagy markers ULK1, FIP200, Atg101, Beclin-1, Atg5, LC3A/B, and cleavage of caspase-3, an indicator of apoptosis in several PDAC cells. ULK1 agonist LYN-1604 enhanced the autophagic and apoptotic effects of PVT. On the other hand, autophagy inhibitors chloroquine and bafilomycin blocked the autophagic and apoptotic effects of PVT in PDAC cells. Notably, chloroquine abrogated the growth suppressive effects of PVT by 25% in BxPC3 tumor xenografts in nude mice. Collectively, our results indicate that PVT mediated pancreatic tumor growth suppression was associated with induction of autophagy mediated apoptosis.

Keywords: apoptosis; autophagy; cancer; cell death; drug repurposing.

Figure 1

PVT treatment increases autophagy in PDAC cells. Autophagic effects of PVT in (A) AsPC1, (B) BxPC3, (C) L3.6pl, (D) MIAPaCa2, and (E) PO2 cells were evaluated by acridine orange assay using flow cytometry. All experiments were independently repeated for 3 times. Data were quantitated using FlowJo software and plotted using GraphPad Prism 7.0. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001.

Figure 2

PVT treatment induces autophagosome formation, lysosomal acidification, and fusion of autophagosome with lysosome. (A) Autophagy induction by PVT treatment was confirmed by confocal microscopy in AsPC1 cells. (B) Localization of acidic lysosomes was confirmed by LysoTracker assay in AsPC1 cells treated with 7.5 µM PVT. (C) Electron microscopic images of control and PVT treated AsPC1 cells showing effect of PVT on autophagosome formation and autophagosome—lysosome fusion. Statistically significant when compared with that of control.

Figure 3

Expression of ULK1 in PDAC patient samples and its effect on overall survival rate. Basal expression of ULK1 in pancreatic tumor patients was evaluated by Cancer Genomic databases (A) R2 and (B) GEPIA. Data are represented as fold change with respect to healthy tissues. Role of ULK1 in (C) Overall patient survival was assessed using GEPIA database. * p ≤ 0.05.

Figure 4

PVT elevates ULK1 mediated autophagy in PDAC cells. Western blot analysis of pULK1(S757), ULK1, Atg101, FIP200, Beclin-1, Atg5, and LC3A/B in (A) PO2 (B) BxPC3 (C) L3.6pl, (D) AsPC1, and (F) MIAPaCa2 cells treated with 2.5, 5 and 7.5 µM PVT for 24 h. β-actin was used as a loading control. Figures shown are representative blots of three independent experiments. Blots were developed using Optimax X-ray film processor (Protec, Germany). PVT enhanced LC3 II/I ratio in (E) AsPC1 and (G) MIAPaCa2 cells treated with PVT for 24 h. Proteins were probed for LC3 antibodies and LC3 II/I band density was measured using Image J. (H) Quantitated representation of ULK1 expression data obtained from Figure 4A–D,F. * p ≤ 0.05, ** p ≤ 0.01.

Figure 5

Confocal microscopy confirms autophagy induction in PVT treated PDAC cells. Immunofluorescence analysis of (A) ULK1 and (B) LC3B (FITC—green), Nucleus (DAPI—blue), Phalloidin (TRITC—Red) in AsPC1 cells treated with 7.5 µM PVT for 24 h.

Figure 6

ULK1 agonist LYN-1604 potentiates autophagic effects of PVT in (A) AsPC1 and (B) PO2 PDAC cells pretreated with 10 µM LYN-1604 for 3 h and then treated with 7.5 µM PVT for 24 h. Blocking autophagy using Bafilomycin suppressed autophagy inducing effects of PVT in (C) AsPC1 (D) BxPC3 PDAC cells pretreated with 10 nM bafilomycin (Baf) for 3 h and then treated with 7.5 µM PVT for 24 h. Single cell suspension was then analyzed by acridine orange assay using flow-cytometry. (E) Western blot analysis of LC3A/B on AsPC1 cells pretreated with 10 nM bafilomycin (Baf) for 3 h and then treated with 7.5 µM PVT for 24 h (F) LC3 II/I ratio from Figure 6E. (G) Western blot analysis of LC3A/B on AsPC1 cells pretreated with 10 µM LYN-1604 for 3 h and then treated with 7.5 µM PVT for 24 h. (H) LC3 II/I ratio from Figure 6G. * p ≤ 0.05, ** p ≤ 0.01.

Figure 7

Anti-proliferative and apoptotic effects of PVT in PDAC cells (A) Cytotoxic effects of PVT were evaluated in L3.6pl cells by SRB assay. (B) Colony formation assay on PO2 cells. (C) AsPC1 (D) BxPC3 (E) L3.6pl and (F) PO2 cells were evaluated by AnnexinV/APC assay. Early- and late-stage apoptosis was added and plotted in graphs. Data were quantitated using FlowJo software and plotted using GraphPad Prism 7.0. Whole cell lysates of (G) AsPC1, BxPC3, L3.6pl, MIAPaCa2, and PO2 cells treated with increasing concentrations of PVT were immunoblotted for cleaved caspase 3. Statistically significant when compared with control. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Figure 8

Modulating autophagy enhances or inhibits the apoptotic effects of PVT in BxPC3 cells. Western blot analysis on AsPC1 or BxPC3 whole cell lysates pretreated with (A,C) 10 µM LYN-1604 (B) LC3B siRNA (E) 10 nM bafilomycin (Baf) for 3 h and then treated with 7.5 µM PVT for 24 h. (D) Quantitated blots from Figure 8C. * p ≤ 0.05.

Figure 9

PVT suppresses growth of BxPC3 tumor xenografts by autophagy-mediated apoptosis. (A) Growth suppressive effects of PVT in subcutaneous PDAC xenograft model (Control, CQ, PVT, CQ + PVT (n = 6)). (B) Representative tumor images. (C) Tumors were excised and the weight was compared between control, chloroquine (CQ), PVT, and chloroquine + PVT (CQ + PVT) groups. (D) Western blot analysis of ULK1, FIP200, LC3A/B and cleaved caspase 3 in BxPC3 tumor lysates. β-actin was used as a loading control. Protein expression in control and treatment groups were normalized with its respective β-actin. Each band represents tumor from individual mouse. (E) ULK1 expression determined by IHC in excised tumors. Statistically significant. p ≤ 0.05. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.