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Eukaryotic cells, when exposed to stress, stop protein translation and form cytoplasmic granules called stress granules (SGs) containing mRNAs and mRNA-binding proteins such as Ras-GAP SH3 domain-binding protein (G3BP) 1 and 2. Cells are thought to survive stress by utilizing SGs as the platform of RNA triage. Accordingly, in cancer cells, SGs are proposed to cause resistance against anti-cancer drugs. We initiated this study to identify novel compounds that induce SGs. We exposed human keratinocyte HaCaT cells to various chemical compounds and immunostained them with anti-G3BP antibody. We found that the cells exposed to topoisomerase inhibitors, teniposide and topotecan, form G3BP-containing aggregates (GCAs). GCAs look like SGs, but are formed more slowly and are not dissociated after stress removal. GCA formation is not associated with eIF2α phosphorylation, while in most cases SG formation depends on it. More importantly, GCAs do not contain representative components of SGs or mRNAs. In sum, we have unexpectedly found a novel cytoplasmic structure ｃontaining G3BP that is distinct from SG.

In eukaryotic cells, when exposed to certain types of stress including hypoxia, eIF2α is phosphorylated by several kinases including protein kinase R (PKR) and PKR-like endoplasmic reticulum kinase (PERK). Subsequently, protein translation is stopped and stress granules (SGs) are formed. Cancer cells form SGs under hypoxia. SGs accumulate apoptosis-related molecules and play anti-apoptotic roles. Thus, hypoxia-induced SG formation contributes to drug resistance in cancer cells. For this reason, inhibition of SG formation is expected to be beneficial in cancer therapy. To prove this concept, chemical reagents that inhibit SG formation are required as experimental tools. We searched for chemical compounds that suppress SG formation and identified that β-estradiol, progesterone, and stanolone (hereafter described as EPS) inhibit SG formation in human cervical cancer HeLa cells. As it turned out, EPS block PKR but not PERK, thus fail to suppress SG formation in most cancer cells, where SGs are formed via PERK. Nevertheless, in this study, we used HeLa cells as a model and demonstrated that EPS block hypoxia-induced SG formation in HeLa cells and consequently reduce drug resistance that HeLa cells acquire under hypoxia. Our findings support that inhibition of SG formation is a useful method to control cancers.