Andrographis paniculata is widely used in traditional herbal medicines for the treatment of common cold, fever and diarrhea, in many regions of Scandinavia and Asia, including Thailand. The pharmacological activities of A. paniculata are mainly attributed to active diterpenoids including 14-deoxyandrographolide, which is uniquely high in first true leaf ethanolic extract (FTLEE) of A. paniculata. In this study, the acute toxicity of the standardized FTLEE of A. paniculata was examined according to the OECD test guideline No. 420. Mice were divided into four groups of each sex and orally received the standardized FTLEE of A. paniculata (0, 300, 2000, or 5000 mg/kg BW). Post-treatment, body weight, signs of toxicity, and/or mortality were observed for 14 days. At Day 15, animals were euthanized, internal organs were observed grossly, and blood samples collected were subjected to hematology and clinical biochemistry analyses. The results showed that all treated animals survived and no apparent adverse effects were observed during the duration of the study. Gross necropsy observation revealed no lesion in any organ of all the standardized FTLEE-treated mice. Although significant alterations in BUN, lymphocytes, neutrophils, hematocrit and hemoglobin were observed, these alterations were not treatment-related toxic effects. Therefore, we concluded that a single oral administration of the standardized FTLEE of A. paniculata with an upper fixed dose of 5000 mg/kg BW has no significant acute toxicological effects.

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 Ca²⁺-dependent cell death. The ATP-generating pyruvate kinase-M2 (PKM2) is oncogenic and overexpressed in PDAC. This study investigated the PKM2-derived 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 membrane-associated proteins. Fura-2 imaging was used to assess cytosolic Ca²⁺ overload and in situ Ca²⁺ 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 Ca²⁺ 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.

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-ADP-ribose-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.

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.

Corneal epithelium, the outmost layer of the cornea, comprises corneal epithelial cells (CECs) that are continuously renewed by limbal epithelial stem cells (LESCs). Loss or dysfunction of LESCs causes limbal stem cell deficiency (LSCD) which results in corneal epithelial integrity loss and visual impairment. To regenerate the ocular surface, transplantation of stem cell-derived CECs is necessary. Human Wharton’s jelly derived mesenchymal stem cells (WJ-MSCs) are a good candidate for cellular therapies in allogeneic transplantation. This study aimed to test the effects of treatments on three signaling pathways involved in CEC differentiation as well as examine the optimal protocol for inducing corneal epithelial differentiation of human WJ-MSCs. All-trans retinoic acid (RA, 5 or 10 µM) inhibited the Wnt signaling pathway via suppressing the translocation of β-catenin from the cytoplasm into the nucleus. SB505124 downregulated the TGF-β signaling pathway via reducing phosphorylation of Smad2. BMP4 did not increase phosphorylation of Smad1/5/8 that is involved in BMP signaling. The combination of RA, SB505124, BMP4, and EGF for the first 3 days of differentiation followed by supplementing hormonal epidermal medium for an additional 6 days could generate corneal epithelial-like cells that expressed a CEC specific marker CK12. This study reveals that WJ-MSCs have the potential to transdifferentiate into CECs which would be beneficial for further applications in LSCD treatment therapy.

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.