Abstract
Cancer cells have been characterized with alkaline intracellular pH (pHi) values (≥7.2) to enable cancer proliferation, migration, and progression. The aim of the present study was to explore the concentration-dependent effects of Andrographolide, an active diterpenoid compound of herb Andrographis paniculata, on Na+/H+ exchanger isoform 1 (NHE1), cellular migration and apoptosis in human cervical cancer cells (HeLa). The pHi was detected by microspectrofluorometry method, and intracellular acidification was induced by NH4Cl prepulse technique. Viability and protein expression were determined by MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and Western blot, respectively. Human normal endocervical cells (End1), ectocervical cells (Ect1), and HeLa were bought commercially. The resting pHi value of HeLa (≈7.47) was significantly higher than that of End1 and Ect1 (≈7.30), and shifted from alkaline to acidic following acid/base impacts. In HEPES (4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid | N-(2-Hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) -buffered superfusate, NHE1 and V-ATPase co-existed functionally for acid extrusion in HeLa, while only NHE1 existed functionally in End/Ect1. Andrographolide (3–1000 μM) concentration-dependently inhibited NHE1 activity. Cell-migration and expressions of NHE1, V-ATPase, PARP (poly-ADP-ribose-polymerase), pro-Caspase-3, and Bcl-2 were significantly reduced by pretreating with Andrographolide (≥100 μM) for 24–48 h in HeLa. Andrographolide inhibited cell viability of End1-cells/Ect1 and HeLa (≥100 and ≥30 μM, respectively). The present findings implicate the promising clinical applications of Andrographolide on cervical cancer treatment.
Highlights
The intracellular pH in most mature mammalian cells is kept within a narrow range (≈7.2)through the combined operation of the active transmembrane transporters and the passive intracellular buffering capacity (β) [1]
Our present study showed that the expression of Na+ /H+ exchanger isoform 1 (NHE1), Bcl-2, PARP, pro-Caspase 3, and cleaved Caspase 3 was significantly reduced by treating with 100 μM
Andrographolide for 24 and 48 h in human cervical cancer cells (HeLa) cells (n = 4, * p < 0.05 or ** p < 0.005), as shown in the with 100 μM Andrographolide for 24 and 48 h in HeLa cells (n = 4, * p < 0.05 or ** p < 0.005), as histograms of Figure 7B–E, respectively. These results suggested that the inhibition of cell migration shown in the by Andrographolide was mainly due to the decreasing functional activity and/or downregulation of protein expression of NHE1, and that the activation of apoptotic pathway involved with Bcl-2, PARP, and Caspase 3 played a key role on the mechanism of Andrographolide-induced anti-cancer effect
Summary
The intracellular pH (pHi ) in most mature mammalian cells is kept within a narrow range (≈7.2)through the combined operation of the active transmembrane transporters and the passive intracellular buffering capacity (β) [1]. Homeostasis of pHi regulates many cellular functions, such as cell growth, migration, and apoptosis in mammalian cells. Some cancer cells have been found recently with alkaline pHi values (≥7.2) and acidic pHe values (≤7.0) [2,3,4]. This “reversed” gradient enables cancer progression by promoting proliferation, evasion of apoptosis, migration, and invasion [5,6]. Maintenance of stable, mildly alkaline pHi by activating some acid extruding mechanisms is required for cancer cell proliferation and differentiation [5,6]. The pHi homeostasis is vital for the cancer development and progression
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