Introduction
Yessotoxin (YTX) and its analogs are a group of lipophilic marine toxins mainly produced by the dinoflagellates Protoceratium reticulatum, Gonyaulax spinifera and Lingulodinium polyedrum. These toxins tend to accumulate in filter-feeding molluscs, have been found in numerous countries worldwide (1–6) and were first detected in shellfish samples collected from Chinese coastal areas in 2009 (7).
YTX and its analogs were first isolated from Patinopecten yessoensis in Japan (8) and were long classified as one of the categories of diarrheic shellfish poisoning (DSP) toxins (9). However, as these toxins cannot induce diarrhea, nor inhibit protein phosphatase 2A, like other DSP toxins, they were classified as an independent group by the European Union Commission in 2002 (10).
Apoptosis is a programmed form of cell suicide. The process of apoptosis is controlled by genes and is crucial in disease outbreaks, including cancer. Once the signaling pathway of apoptosis is activated, the process cannot be easily undergone, even by tumor cells. Thus, there is an increasing number of studies on tumor cell apoptosis, with the aim to design an effective cancer treatment (11–13). Several studies indicated that YTX may induce apoptosis in different types of cell lines (14–21). However, the exact underlying mechanisms have not been elucidated.
As the chemical structure of YTX is similar to that of brevetoxins and ciguatoxins, which were shown to interfere with voltage-gated sodium channels, the effects of YTX on transmembrane ion channels were previously investigated (22,23). The present study mainly investigated the YTX-induced alterations in intracellular Ca2+ levels in Bel7402 human hepatocellular carcinoma cells and the possible underlying mechanisms.
Materials and methods
Reagents
Pure YTX was purchased from the National Research Council (NRC; Ottawa, ON, Canada). Fluo-3 acetoxymethyl ester (AM) solution (5 mM) was purchased from Beyotime Institute of Biotechnology (Shanghai, China). Nifedipine was purchased from Sigma (St. Louis, MO, USA) and was dissolved in dimethyl sulfoxide (Merck KGaA, Darmstadt, Germany). All other chemicals were of analytical reagent grade or higher purity.
The Hanks’ balanced salt solution (HBSS) used for cell washing consisted of 137 mM NaCl, 5.6 mM KCl, 1.26 mM CaCl2, 0.81 mM MgSO4, 0.38 mM Na2HPO4, 0.44 mM KH2PO4 and 4.2 mM NaHCO3. The pH of the HBSS was adjusted to 7.4 with 0.1 M HCl and NaOH.
Cell culture
The Bel7402 human hepatocellular carcinoma cell line was purchased from the Cell Bank of the Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China). The cells were cultured in RPMI-1640 medium (Gibco, Carlsbad, CA, USA) supplemented with 300 mg/l L-glutamine, 100 μg/ml streptomycin and 100 U/ml penicillin, plus 10% fetal calf serum (TBD, Beijing, China) and incubated at 37°C in a humidified 5% CO2 atmosphere.
Analysis of intracellular Ca<sup>2+</sup> levels
The Bel7402 cells were transferred to a 24-well microplate and incubated at 37°C. When 90% confluence was reached, the cells were washed twice with HBSS and loaded with Fluo-3 AM at a final concentration of 5 μM for 1 h at 37°C in the dark. The solution containing the dye was then removed and the cells were washed twice with HBSS.
The average fluorescence intensity of intracellular Ca2+ concentration in labeled cells was detected under a laser scanning confocal microscope (FluoView FV1000; Olympus Co., Tokyo, Japan). The wavelength of excitation was set at 488 nm and the emission wavelength at 525 nm for Fluo-3 fluorescence reading. Newly-developed FV300/FV500 Application software was used for the measurement and analysis of Ca2+ concentration.
Ethylene glycol tetraacetic acid (EGTA) and nifedipine, at a final concentration of 2 mM and 1 μM, respectively, were added to the reaction system to investigate the exact mechanism of YTX-evoked Ca2+ increase.
Discussion
YTX is a low-molecular weight compound that is toxic to different mammalian and insect cell types, including cancer cells (14,15,23–26). Our previous studies demonstrated the apoptosis induced by YTX in HeLa human cervical cancer cells, Bel7402 human hepatocellular carcinoma cells and HL7702 human liver cells (19–21). The identification of cell death signalling pathways and cellular factors involved in apoptosis induced by YTX may provide useful information for evaluating the potential use of YTX for therapeutic purposes, including cancer research. Botana Lopez et al(27) indicated that YTX may be used as an antitumor agent, with this possibility already considered in the European patent application EP1875906.
Apoptosis, the process of programmed cell death, is often characterized by an orderly progression of morphological changes, including cell shrinkage, condensation of chromatin and externalization of membrane phosphatidylserine, and is controlled by cell signals that may originate extracellularly or intracellularly (28). These signals may positively or negatively affect apoptosis. Extracellular signals, including toxins, hormones, growth factors, nitric oxide and cytokines (29), may either cross the plasma membrane or transduce into intracellular signals in order to affect other responses. Ca2+ is an important intracellular second messenger which is crucial in numerous processes, including cell proliferation, differentiation, DNA damage and apoptosis (30,31). Thus, the changes in the cytoplasmic Ca2+ concentration may induce the process of apoptosis (32,33). Previous studies indicated that YTX may induce the increase of Ca2+ levels in several types of cells (22,23,26). Our previous studies also demonstrated that YTX may induce Ca2+ entry in different types of human cells (21,34). Ca2+ ions are crucial in the process of apoptosis and cytoplasmic Ca2+ concentration is involved in the modulation of several cell functions. It was previously suggested that the capacitative Ca2+ influx through Ca2+ channels and the Ca2+ released in cytoplasm from intracellular organelles are apoptogenic (35–37). As described by Putney (38,39), the release of Ca2+ from intercellular stores may induce Ca2+ influx from the extracellular medium. However, de la Rosa et al(22) demonstrated that the Ca2+ influx induced by YTX was not affected by internal stores. Our previous study also indicated that YTX did not affect internal Ca2+ levels in a Ca2+-free medium (21).
Although the changes in the Ca2+ levels may be associated with the apoptosis induced by YTX, the exact mechanism underlying this increase in Ca2+ has not been elucidated. The chemical structure of YTX is similar to that of brevetoxins, which are known to interfere with sodium channel function. Therefore, it is possible that YTX is also able to interact with cellular ion channels. We previously demonstrated that nifedipine, a specific L-type Ca2+ channel blocker, was able to inhibit the Ca2+ increase induced by YTX in several human cell lines (21,34). YTX may exert an effect on L-type Ca2+ channel in cells. Although YTX may induce the Ca2+ increase in HL7702 normal human liver cells and Bel7402 human hepatocellular carcinoma cells, the increasing trend of Ca2+ levels in Bel7402 cells was different from that observed in HL7702 cells (34). This difference may provide new insight into cancer therapy. Further studies are required to elucidate the exact mechanisms underlying apoptosis induced by YTX in tumor cells.
In conclusion, YTX was shown to exert a cytotoxic effect on normal human liver cells and human hepatocellular carcinoma cells. Although YTX may induce Ca2+ influx in these two cell types, the detailed increasing trend of Ca2+ levels was different. The difference between normal human liver cells and human hepatocellular carcinoma cells may provide new insight into cancer therapy.