H2O2 Signature and Innate Antioxidative Profile Make the Difference Between Sensitivity and Tolerance to Salt in Rice Cells.

Salt tolerance is a complex trait that varies between and within species. H2O2 profiles as well as antioxidative systems have been investigated in the cultured cells of rice obtained from Italian rice varieties with different salt tolerance. Salt stress highlighted differences in extracellular and intracellular H2O2 profiles in the two cell cultures. The tolerant variety had innate reactive oxygen species (ROS) scavenging systems that enabled ROS, in particular H2O2, to act as a signal molecule rather than a damaging one. Different intracellular H2O2 profiles were also observed: in tolerant cells, an early and narrow peak was detected at 5 min; while in sensitive cells, a large peak was associated with cell death. Likewise, the transcription factor salt-responsive ethylene responsive factor 1 (TF SERF1), which is known for being regulated by H2O2, showed a different expression profile in the two cell lines. Notably, similar H2O2 profiles and cell fates were also obtained when exogenous H2O2 was produced by glucose/glucose oxidase (GOX) treatment. Under salt stress, the tolerant variety also exhibited rapid upregulation of K+ transporter genes in order to deal with K+/Na+ impairment. This upregulation was not detected in the presence of oxidative stress alone. The importance of the innate antioxidative profile was confirmed by the protective effect of experimentally increased glutathione in salt-treated sensitive cells. Overall, these results underline the importance of specific H2O2 signatures and innate antioxidative systems in modulating ionic and redox homeostasis for salt stress tolerance.

Plant Cell Cultures as Model Systems to Study Programmed Cell Death.

The study of programmed cell death (PCD) activated in a certain group of cells is complex when analyzed in the whole plant. Plant cell suspension cultures are useful when investigating PCD triggered by environmental and developmental stimuli. Due to their homogeneity and the possibility to synchronize their responses induced by external stimuli, these cultures are used for studying the signaling pathways leading to PCD. The first problem in the analysis of PCD in cell cultures is the quantification of cell viability/death over time. Cultured cells from different plant species may have specific mitotic patterns leading to calli or cell chains mixed to single cell suspensions. For this reason, not all cell cultures allow morphological parameters to be investigated using microscopy analysis, and adapted or ad hoc methods are needed to test cell viability.Here we report on some accurate methods to establish and propagate cell cultures from different plant species, including crops, as well as to determine cell viability and PCD morphological and genetic markers. In particular, we describe a protocol for extracting nucleic acids required for real-time PCR analysis which has been optimized for those cell cultures that do not allow the use of commercial kits.

Tocotrienols induce endoplasmic reticulum stress and apoptosis in cervical cancer cells.

BACKGROUND: We have previously reported that γ- and δ-tocotrienols (γ- and δ-T3) induce gene expression and apoptosis in human breast cancer cells (MDA-MB-231 and MCF-7). This effect is mediated, at least in part, by a specific binding and activation of the estrogen receptor-ß (ERß). Transcriptomic data obtained within our previous studies, interrogated by different bioinformatic tools, suggested the existence of an alternative pathway, activated by specific T3 forms and leading to apoptosis, also in tumor cells not expressing ER. In order to confirm this hypothesis, we conducted a study in HeLa cells, a line of human cervical cancer cells void of any canonical ER form. RESULTS: Cells were synchronized by starvation and treated either with a T3-rich fraction from palm oil (10-20 µg/ml) or with purified α-, γ-, and δ-T3 (5-20 µg/ml). α-tocopherol (TOC) was utilized as a negative control. Apoptosis, accompanied by a significant expression of caspase 8, caspase 10, and caspase 12 was observed at 12 h from treatments. The interrogation of data obtained from transcriptomic platforms (NuGO Affymetrix Human Genechip NuGO_Hs1a520180), further confirmed by RT-PCR, suggested that the administration of γ- and δ-T3 associates with Ca2+ release. Data interrogation were confirmed in living cells; in fact, Ca-dependent signals were observed followed by the expression and activation of IRE-1α and of other molecules involved in the unfolded protein response, the core pathway coping with endoplasmic reticulum stress in eukaryotic cells, finally leading to apoptosis. CONCLUSIONS: Our study demonstrates that γ- and δ-T3 induce apoptosis also in tumor cells lacking of ERß by triggering signals originating from endoplasmic reticulum stress. Our observations suggest that tocotrienols could have a significant role in tumor cell physiology and a possible therapeutic potential.