PTEN inhibition leads to the development of resistance to novel isoquinoline derivative TNBG-5602 in human liver cancer cells.

TNBG-5602, a new synthesized derivative of tetrazanbigen, is a potential chemotherapeutic agent against cancer. However, its underlying mechanism is complex and still unknown. In this investigation, the anticancer effects of TNBG-5602 were determined in vitro and in vivo. Small RNA retroviral library plasmids that overexpress 19-bp fragments were used to generate TNBG-5602-resistant cells. After validation, the overexpressed 19-bp fragments were sequenced using next-generation sequencing (NGS) in the drug-resistant cells. Furthermore, the relationship of TNBG-5602, phosphatase and tensin homolog deleted on Chromosome 10 (PTEN), and the phosphatidylinositol 3 kinase (PI3K)/protein kinase B (Akt) pathway was explored. The results showed that TNBG-5602 can effectively inhibit cancer cell proliferation and induce apoptosis in vitro and in vivo. Drug-resistant cells were screened using the small RNA library. Compared with naïve cells, drug-resistant cells were more resistant to TNBG-5602 in vitro and in vivo. NGS results revealed that the second highest overexpressed 19-bp fragment perfectly matched the PTEN gene, so the expression of PTEN in various cells and tissues was verified. Further research showed that exogenous overexpression of PTEN strengthened the anticancer effects of TNBG-5602 on p-Akt expression, whereas silencing of PTEN weakened these effects in naïve cells. Taken together, by using this library, we confirmed that PTEN is the target gene to the anticancer effects of TNBG-5602 via the PI3K/Akt pathway.

A homologue of vitamin K epoxide reductase in Solanum lycopersicum is involved in resistance to osmotic stress.

Vitamin K epoxide reductase (VKOR) exists widely in the chloroplasts of higher plants and plays important roles in redox regulation. However, investigations of plant VKOR function have primarily focused on VKOR from Arabidopsis, and knowledge concerning this function is therefore quite limited. In this study, Solanum lycopersicum was used to study the function of VKOR. Transcription level analysis revealed that SlVKOR (S. lycopersicum VKOR) expression was upregulated by salt or drought stress. To further investigate the function of SlVKOR in plants, we generated sense and antisense transgenic S. lycopersicum homozygotes at T2 generation plants. Compared with wild-type (WT) plants, the leaf disks of the SlVKOR overexpression plants retained a much higher percentage of chlorophyll after salt or drought treatment, whereas the antisense transgenic plants displayed an opposite response. The overexpressed plants displayed lower levels of H2O2 and superoxide anion radical (O2(·-)) than WT plants, whereas antisense plants accumulated considerably more H2O2 and O2(·-). The activities of reactive oxygen scavenger enzymes, including superoxide dismutase, peroxidase, ascorbate peroxidase and catalase, were consistent with the accumulation of reactive oxygen species. Based on these results, we suggest that SlVKOR is involved in resistance to salt or drought stress.

Identification and characterization of SlVKOR, a disulfide bond formation protein from Solanum lycopersicum, and bioinformatic analysis of plant VKORs.

Homologs of vitamin K epoxide reductase (VKOR) exist widely in plants. However, only VKOR of Arabidopsis thaliana has been the subject of many studies to date. In the present study, the coding region of a VKOR from Solanum lycopersicum (JF951971 in GenBank) was cloned; it contained a membrane domain (VKOR domain) and an additional soluble thioredoxin-like (Trx-like) domain. Bioinformatic analysis showed that the first 47 amino acids in the N-terminus should act as a transit peptide targeting the protein to the chloroplast. Western blot demonstrated that the protein is localized in thylakoid membrane with the Trx-like domain facing the lumen. Modeling of three-dimensional structure showed that SlVKOR has a similar conformation with Arabidopsis and cyanobacterial VKORs, with five transmembrane segments in the VKOR domain and a typical Trx-like domain in the lumen. Functional assay showed that the full-length of SlVKOR with Trx-like domain without the transit peptide could catalyze the formation of disulfide bonds. Similar transit peptides at the N-terminus commonly exist in plant VKORs, most of them targeting to chloroplast according to prediction. Comparison of sequences and structures from different plants indicated that all plant VKORs possess two domains, a transmembrane VKOR domain and a soluble Trx-like domain, each having four conservative cysteines. The cysteines were predicted to be related to the function of catalyzing the formation of disulfide bonds.