Rlz-8 Induces An Autophagic Cell Death In Human Gastric Cancer SGC-7901 Cells

In recent years, the relationship between the intracellular aggregation of unfolded or misfolded proteins and ER stress has been intensively examined. However, there are few reports in the cell biological literature regarding ER stress and autophagy induced by a heterologous protein. The UPR system is composed of two ER-associated degradation (ERAD) systems, the ubiquitin/proteasome and the autophagy/lysosome. The UPR system is composed of two ER-associated degradation (ERAD) systems, the ubiquitin/proteasome and the autophagy/lysosome.Ganoderma lucidum, a popular medicinal mushroom, has been widely used in traditional Chinese medicine in many Asian countries during the past two millennia. It has been reported to be effective in modulating immune functions, inhibiting tumor cell growth and allergies and in the treatment of chronic hepatitis, hypertension, and hyperglycemia. G. lucidum polysaccharide and triterperoid were the major bioactive substances until immunomodulatory proteins, Lz-8, was isolated and purified from the mycelia of G. lucidum in 1989. Lz-8 was a noncovalently linked homodimer with an apparent molecular weight of 24 kDa. Each monomer consists of 110 aa residues with an acetylated N terminus and a molecular mass of 12 kDa. In a previous study, LZ-8 was recombinantly expressed in Pichia pastoris, and we demonstrated for the first time that the protein structure of recombinant fungal immunomodulatory protein Ling Zhi-8 (rlz-8). It was also reported that reFIP-gts, another immunomodulatory protein from the closely related Ganoderma tsugae, inhibited the growth of A549 cancer cells significantly and selectively. But until now there have been no studies demonstrating how Lz-8 induces cell death and the mechanisms involved in this process.Disturbances in normal functions of the ER lead to the UPR, an evolutionarily conserved cell stress response which is aimed initially at compensating for damage but can eventually trigger cell death if ER dysfunction is severe or prolonged. The mechanisms by which ER stress leads to cell death remain enigmatic, with multiple potential participants described with little clarity, with specific death effectors dominating in particular cellular contexts. As we know, types of cell death induced by ER stress include apoptotic cell death, necrosis, and autophagic cell death. Among these, apoptotic cell death depends greatly on caspase activity. Many important relevant mechanisms involved in caspase-dependent cell death have already been studied extensively.In our study, rlz-8 exerted a promising killing effect on SGC-7901 cells. We used classical methods to examine whether caspase-independent cell death rather than caspase-dependent apoptosis was triggered by rlz-8-induced ER stress. The results of TEM indicated that no morphological feature of caspase-dependent apoptosis was observed in rlz-8-treated cells, i.e., chromatin condensation, loss of cytoplasmic processes, round cell shape or fading of the nuclear membrane. Moreover, when caspase families were inhibited with the pan-caspase inhibitor z-VAD-FMK in SGC-7901 cells, we found that rlz-8 still had the same killing effect, affirming that rlz-8-induced cell death is not caspase-dependent, and may not even be apoptosis.Regarding rlz-8-induced cell death, our findings suggest:(1) In SGC-7901 cells, rlz-8 diffuses and accumulates gradually in the ER, reaching saturation after 24 h. rlz-8 was not transported out of the ER or degraded until the cells died. (2) After rlz-8 accumulated in the ER, a significant increase in the number of lysosomes was observed, by fluorescence microscopy. The results of TEM also confirmed dilated and vacuolized smooth ER and an increased number of lysosomes. (3) rlz-8-treated cells showed increased mRNA and protein levels of CHOP/GADD153 and ATF4, respectively. (4) When rlz-8 gradually accumulated in the ER of SGC-7901 cells, autophagy and cell death were observed. When autophagy was inhibited by 3-MA treatment, the number of dead cells decreased, suggesting that autophagy played a role in promoting cell death in rlz-8 treated cells.Therefore, how does the cell deal with the aggregation of rlz-8 in the ER? Excess amounts of unfolded or misfolded proteins in the ER are retrotranslocated to the cytoplasm and degraded by the ERAD system, which includes an ubiquitine/proteasome and an autophagy/lysosome system. A larger number of autophagosomes and lysosomes appeared in rlz-8-treated cells. This suggests that the autophagy/lysosome system was activated, and the cell may employ this system to degrade rlz-8. Although there were no experimental results in our study that verified that rlz-8 was recognized and degraded by the ubiquitin/proteasome ERAD system, we used a Bayesian discriminant method to predict rlz-8's ubiquitination sites. However, specific ubiquitination sites were not found.In general, the ERAD system degrades aberrant proteins to ensure cell survival, but in this study the SGC-7901 cells were killed by the accumulation of rlz-8 in the ER. Similar studies also indicated that autophagy protects cells from ER stress arising from protein aggregations via the ERAD system. However, we have determined that while aggregations of rlz-8 induced ER stress and triggered the ERAD system, the resulting autophagy contributed to cell death rather than survival. Before this, it was very rare that a heterologous protein aggregating in the ER of a tumor cell could trigger an autophagy-dependent cell death.Next, what role did ER stress play in the rlz-8-induced cell death? As we know, when ER stress exceeds a threshold it mediates many types of cell death, including classic apoptosis, autophagic cell death and necrosis. The principal challenge with any cellular strategy for preventing death caused by ER stress lies with the multitude of parallel pathways that potentially lead to downstream cell death mechanisms. Multiple pathways linking ER stress to cell death have been reported. Of these, ATF4-CHOP/GADD153 is the first identified protein that mediates ER stress-induced apoptosis and much is known of the roles of this molecule in cell death. Nevertheless, little is known of just how CHOP/GADD153 induces cell death, especially considering that CHOP-deficient cells are resistant to ER stress-mediated cell death. Our study showed that when expression of ATF4 or CHOP was inhibited in rlz-8-treated SGC-7901 cells, dead cells decreased significantly, and LC3B conversion was down-regulated. This proves that the ATF4-CHOP pathway and ER-stress both are the key factors for inducing cell death, and also that the ATF4-CHOP pathway acts as a bridge between ER stress and autophagy.Here, we have investigated that the link between rlz-8 induced cell death. ER stress, autophagy in the rlz-8 treated Human gastric cancer SGC-7901 cells. We have proven that rlz-8 induced cell death is mediated by ER stress, not a classical apoptosis, also autophagy induced by ER stress promotes cell death to a great extent in SGC-7901 cells.In conclusion, rlz-8 aggregation induces autophagic cell death and provides a novel mechanism for protein-based anti-cancer medicine. This may lead to new strategies to develop therapeutic drugs that will target cancer cells to undergo autophagic cell death independent of apoptosis.