Pomegranate extract inhibits migration and invasion of oral cancer cells by downregulating matrix metalloproteinase-2/9 and epithelial-mesenchymal transition.

Discovering drug candidates for the modulation of metastasis is of great importance in inhibiting oral cancer malignancy. Although most pomegranate extract applications aim at the antiproliferation of cancer cells, its antimetastatic effects remain unclear, especially for oral cancer cells. The aim of this study is to evaluate the change of two main metastasis characters, migration and invasion of oral cancer cells. Further, we want to explore the molecular mechanisms of action of pomegranate extract (POMx) at low cytotoxic concentration. We found that POMx ranged from 0 to 50 µg/mL showing low cytotoxicity to oral cancer cells. In the case of oral cancer HSC-3 and Ca9-22 cells, POMx inhibits wound healing migration, transwell migration, and matrix gel invasion. Mechanistically, POMx downregulates matrix metalloproteinase (MMP)-2 and MMP-9 activities and expressions as well as epithelial-mesenchymal transition (EMT) signaling. POMx upregulates extracellular signal-regulated kinases 1/2 (ERK1/2), but not c-Jun N-terminal kinase (JNK) and p38 expression. Addition of ERK1/2 inhibitor (PD98059) significantly recovered the POMx-suppressed transwell migration and MMP-2/-9 activities in HSC-3 cells. Taken together, these findings suggest to further test low cytotoxic concentrations of POMx as a potential antimetastatic therapy against oral cancer cells.

Ethyl acetate extracts of Nepenthes ventricosa x sibuyanensis leaves cause growth inhibition against oral cancer cells via oxidative stress.

Introduction: The genus Nepenthes of the pitcher plants contains several natural and hybrid species that are commonly used in herbal medicine in several countries, but its possible use in cancer applications remains unknown as yet. Methods: In this study, we investigated the antioral cancer properties using ethyl acetate extracts of the Nepenthes hybrid (Nepenthes ventricosa x sibuyanensis), namely EANS. The bioactivity was detected by a MTS-based cell proliferation assay and flow cytometric or Western blot analysis for apoptosis, oxidative stress, and DNA damage. Results: Treatment for 24 hrs of EANS inhibited all three types of oral cancer cells that were tested (Ca9-22, CAL 27, and SCC9), with just a small difference to normal oral cells (HGF-1). This antiproliferation was inhibited by pretreatments with the reactive oxygen species (ROS) scavenger N-acetylcysteine (NAC), and the apoptosis inhibitor (Z-VAD). EANS treatment increased the subG1 population and it also dose- and time-dependently induced annexin V- and pancaspase-detected apoptosis as well as cleaved caspases 3 and 9 overexpressions in the oral cancer cells (Ca9-22). After EANS treatment of Ca9-22 cells, intracellular ROS and mitochondrial superoxide (MitoSOX) were overexpressed and mitochondrial membrane potential (MMP) was disrupted. Moreover, DNA damages such as γH2AX and 8-oxo-2'-deoxyguanosine (8-oxodG) were increased after EANS treatment to Ca9-22 cells. The EANS-induced effects (namely, oxidative stress, apoptosis, and DNA damage) were suppressed by ROS scavenger. Conclusion: Our findings demonstrated that EANS inhibits ROS-mediated proliferation against oral cancer cells.

Ethyl acetate extract of Nepenthes adrianii x clipeata induces antiproliferation, apoptosis, and DNA damage against oral cancer cells through oxidative stress.

Nepenthes plants are regarded as a kind of Traditional Chinese Medicine for several diseases but its anticancer activity remain unclear. The subject of this study is to evaluate the antiproliferation effects on oral cancer cells by Nepenthes plants using ethyl acetate extract of Nepenthes adrianii x clipeata (EANA). Cell viability was detected using MTS assay. Its detailed mechanisms including cell cycle, apoptosis, oxidative stress, and DNA damage were explored by flow cytometry or western blotting. For 24 hours EANA treatment, five kinds of oral cancer cells (CAL 27, Ca9-22, OECM-1, HSC-3, and SCC9) show IC50 values of cell viability ranging from 8 to 17 µg/mL but the viability of normal oral cells (HGF-1) remains over 80%. Subsequently, CAL 27 and Ca9-22 cells with high sensitivity to EANA were chosen to investigate the detailed mechanism. EANA displays the time course and concentration effects for inducing apoptosis based on flow cytometry (subG1 and annexin V analyses) and western blotting [cleaved poly (ADP-ribose) polymerase (c-PARP)]. Oxidative stress and DNA damage were induced by EANA treatments in oral cancer cells through reactive oxygen species (ROS), mitochondrial membrane potential disruption, mitochondrial superoxide, and γH2AX. All these changes of EANA treatments in oral cancer cells were reverted by the ROS scavenger N-acetylcysteine pretreatment. Therefore, EANA induces preferential killing, apoptosis, and DNA damage against oral cancer cells through oxidative stress.