BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) has poor survival and treatment options. PDAC cells shift their metabolism towards glycolysis, which fuels the plasma membrane calcium pump (PMCA), thereby preventing Ca 2+-dependent cell death. The ATP-generating pyruvate kinase-M2 (PKM2) is oncogenic and overexpressed in PDAC. This study investigated the PKM2derived ATP supply to the PMCA as a potential therapeutic locus. METHODS: PDAC cell growth, migration and death were assessed by using sulforhodamine-B/tetrazolium-based assays, gap closure assay and poly-ADP ribose polymerase (PARP1) cleavage, respectively. Cellular ATP and metabolism were assessed using luciferase/fluorescent-based assays and the Seahorse XFe96 analyzer, respectively. Cell surface biotinylation identified membraneassociated proteins. Fura-2 imaging was used to assess cytosolic Ca 2+ overload and in situ Ca 2+ clearance. PKM2 knockdown was achieved using siRNA. RESULTS: The PKM2 inhibitor (shikonin) reduced PDAC cell proliferation, cell migration and induced cell death. This was due to inhibition of glycolysis, ATP depletion, inhibition of PMCA and cytotoxic Ca 2+ overload. PKM2 associates with plasma membrane proteins providing a privileged ATP supply to the PMCA. PKM2 knockdown reduced PMCA activity and reduced the sensitivity of shikonin-induced cell death. CONCLUSIONS: Cutting off the PKM2-derived ATP supply to the PMCA represents a novel therapeutic strategy for the treatment of PDAC.

Graphical abstract

Pancreatic ductal adenocarcinoma (PDAC) is largely resistant to standard treatments leading to poor patient survival. The expression of plasma membrane calcium ATPase-4 (PMCA4) is reported to modulate key cancer hallmarks including cell migration, growth, and apoptotic resistance. Data-mining revealed that PMCA4 was over-expressed in pancreatic ductal adenocarcinoma (PDAC) tumors which correlated with poor patient survival. Western blot and RT-qPCR revealed that MIA PaCa-2 cells almost exclusively express PMCA4 making these a suitable cellular model of PDAC with poor patient survival. Knockdown of PMCA4 in MIA PaCa-2 cells (using siRNA) reduced cytosolic Ca2+ ([Ca2+]i) clearance, cell migration, and sensitized cells to apoptosis, without affecting cell growth. Knocking down PMCA4 had minimal effects on numerous metabolic parameters (as assessed using the Seahorse XF analyzer). In summary, this study provides the first evidence that PMCA4 is over-expressed in PDAC and plays a role in cell migration and apoptotic resistance in MIA PaCa-2 cells. This suggests that PMCA4 may offer an attractive novel therapeutic target in PDAC.

Background: High glycolytic rate is a hallmark of cancer (Warburg effect). Glycolytic ATP is required for fuelling plasma membrane calcium ATPases (PMCAs), responsible for extrusion of cytosolic calcium, in pancreatic ductal adenocarcinoma (PDAC). Phosphofructokinase-fructose-bisphosphatase-3 (PFKFB3) is a glycolytic driver that activates key rate-limiting enzyme Phosphofructokinase-1; we investigated whether PFKFB3 is required for PMCA function in PDAC cells. Methods: PDAC cell-lines, MIA PaCa-2, BxPC-3, PANC1 and non-cancerous human pancreatic stellate cells (HPSCs) were used. Cell growth, death and metabolism were assessed using sulforhodamine-B/tetrazolium-based assays, poly-ADPribose-polymerase (PARP1) cleavage and seahorse XF analysis, respectively. ATP was measured using a luciferase-based assay, membrane proteins were isolated using a kit and intracellular calcium concentration and PMCA activity were measured using Fura-2 fluorescence imaging. Results: PFKFB3 was highly expressed in PDAC cells but not HPSCs. In MIA PaCa-2, a pool of PFKFB3 was identified at the plasma membrane. PFKFB3 inhibitor, PFK15, caused reduced cell growth and PMCA activity, leading to calcium overload and apoptosis in PDAC cells. PFK15 reduced glycolysis but had no effect on steady-state ATP concentration in MIA PaCa-2. Conclusions: PFKFB3 is important for maintaining PMCA function in PDAC, independently of cytosolic ATP levels and may be involved in providing a localised ATP supply at the plasma membrane.

Hepatocellular carcinoma is the most common type of primary liver cancer in humans. This study aimed to demonstrate anticancer properties of an aqueous extract from Chrysophyllum cainito stem bark (CE) and its underlying mechanisms. Our MTT assay results showed that CE significantly reduced human hepatocellular carcinoma (HepG2) cell viability with the IC50of 100 µg/mL, while human dermal primary fibroblast (HDFa) cells showed less susceptibility in every concentration tested. Determined by Annexin V staining, the proportion of apoptotic HepG2 cells increased in a dose-dependent fashion after 24 hour-exposure of CE. The results from Western blot analysis confirmed that CE reduced procaspase-3, suggesting apoptosis by activating caspase-3 cleavage. Using the DCFH-DA and DiOC6 fluorescent probes, it was found that CE significantly stimulated the generation of reactive oxygen species (ROS) and reduced mitochondrial membrane potential (Δψ m), respectively. According to cell cycle analysis, CE (100 µg/mL) profoundly increased the percentage of cells in the sub-G1 phase, indicating cell apoptosis. These data suggest that CE induces apoptosis and cell death in human hepatocellular carcinoma via generation of intracellular ROS and disruption of Δψm. This is the first demonstration of the anticancer activity with proposed underlying mechanism of CE in liver cancer cells.