GRP78/BiP determines senescence evasion cell fate after cisplatin-based chemotherapy.

Cisplatin (CDDP) induces senescence characterized by senescence-associated secretory phenotypes (SASP) and the unfolded protein response (UPR). In this study, we investigated the proteins related to the UPR during the senescence cell fate. Strikingly, we found that one of the critical ER-resident proteins, GRP78/BiP, was significantly altered. Here we show that GRP78 levels differentially expressed depending on non-small lung cancer subtypes. GRP78 indeed regulates the evasion of senescence in adenocarcinoma A549 cells, in which the increased GRP78 levels enable them to re-proliferate after CDDP removal. Conversely, GRP78 is downregulated in the senescence H460 cells, making them lacking senescence evasion capability. We observed that the translational regulation critically contributed to the GRP78 protein levels in CDDP-induces senescence. Furthermore, the increased GRP78 level during senescence confers resistance to senolytic drug, Bortezomib, as observed by a twofold increase in IC50 in A549 senescence cells compared to the wild-type. This observation is also consistent in the cells that have undergone genetic manipulation by transfection with pcDNA3.1(+)-GRP78/BiP plasmids and pSpCas9(BB)-2A-Puro containing guide RNA sequence targeting GRP78 exon 3 to induce the overexpression and downregulation of GRP78 in H460 cells, respectively. Our findings reveal a unique role of GRP78 on the senescence evasion cell fate and senolytic drug resistance after cisplatin-based chemotherapy.

Peptides extracted from edible mushroom: Lentinus squarrosulus induces apoptosis in human lung cancer cells.

CONTEXT: Lentinus squarrosulus Mont. (Polyporaceae) is an interesting source of diverse bioactive compounds. OBJECTIVE: This is the first study of the anticancer activity and underlying mechanism of peptides extracted from Lentinus squarrosuls. MATERIALS AND METHODS: Peptides were isolated from the aqueous extract of L. squarrosulus by employing solid ammonium sulphate precipitation. They were further purified by ion-exchange chromatography on diethylaminoethanol (DEAE)-cellulose and gel filtration chromatography on Sephadex G25. Anticancer activity was investigated in human lung cancer H460, H292 and H23 cells cultured with 0-40 µg/mL of peptide extracts for 24 h. Cell viability and mode of cell death were evaluated by MTT and nuclear staining assay, respectively. Western blotting was used to investigate the alteration of apoptosis-regulating proteins in lung cancer cells treated with peptide extracts (0-20 µg/mL) for 24 h. RESULTS: The cytotoxicity of partially-purified peptide extracts from L. squarrosulus was indicated with IC50 of ∼26.84 ± 2.84, 2.80 ± 2.14 and 18.84 ± 0.30 µg/mL in lung cancer H460, H292 and H23 cells, respectively. The extracts at 20 µg/mL induced apoptosis through the reduction of anti-apoptotic Bcl-2 protein (∼0.5-fold reduction) and up-regulation of BAX (∼4.5-fold induction), a pro-apoptotic protein. Furthermore, L. squarrosulus peptide extracts (20 µg/mL) also decreased the cellular level of death receptor inhibitor c-FLIP (∼0.6-fold reduction). CONCLUSIONS AND DISCUSSION: This study provides the novel anticancer activity and mechanism of L. squarrosulus peptide extracts, which encourage further investigation and development of the extracts for anticancer use.

Citrate synthase mutants of Agrobacterium are attenuated in virulence and display reduced vir gene induction.

A citrate synthase (CS) deletion mutant of Agrobacterium tumefaciens C58 is highly attenuated in virulence. The identity of the mutant was initially determined from its amino acid sequence, which is 68% identical to Escherichia coli and 77% identical to Brucella melitensis. The mutant lost all CS enzymatic activity, and a cloned CS gene complemented a CS mutation in Sinorhizobium. The CS mutation resulted in a 10-fold reduction in vir gene expression, which likely accounts for the attenuated virulence. When a plasmid containing a constitutive virG [virG(Con)] locus was introduced into this mutant, the level of vir gene induction was restored to nearly wild-type level. Further, the virG(Con)-complemented CS mutant strain induced tumors that were similar in size and number to those induced by the parental strain. The CS mutation resulted in only a minor reduction in growth rate in a glucose-salts medium. Both the CS mutant and the virG(Con)-complemented CS strain displayed similar growth deficiencies in a glucose-salts medium, indicating that the reduced growth rate of the CS mutant could not be responsible for the attenuated virulence. A search of the genome of A. tumefaciens C58 revealed four proteins, encoded on different replicons, with conserved CS motifs. However, only the locus that when mutated resulted in an attenuated phenotype has CS activity. Mutations in the other three loci did not result in attenuated virulence and any loss of CS activity, and none were able to complement the CS mutation in Sinorhizobium. The function of these loci remains unknown.