Serine-arginine protein kinase 1 (SRPK1) promotes EGFR-TKI resistance by enhancing GSK3ß Ser9 autophosphorylation independent of its kinase activity in non-small-cell lung cancer.

Resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) is a major challenge for clinicians and patients with non-small cell lung cancer (NSCLC). Serine-arginine protein kinase 1 (SRPK1) is a key oncoprotein in the EGFR/AKT pathway that participates in tumorigenesis. We found that high SRPK1 expression was significantly associated with poor progression-free survival (PFS) in patients with advanced NSCLC undergoing gefitinib treatment. Both in vitro and in vivo assays suggested that SRPK1 reduced the ability of gefitinib to induce apoptosis in sensitive NSCLC cells independently of its kinase activity. Moreover, SRPK1 facilitated binding between LEF1, ß-catenin and the EGFR promoter region to increase EGFR expression and promote the accumulation and phosphorylation of membrane EGFR. Furthermore, we verified that the SRPK1 spacer domain bound to GSK3ß and enhanced its autophosphorylation at Ser9 to activate the Wnt pathway, thereby promoting the expression of Wnt target genes such as Bcl-X. The correlation between SRPK1 and EGFR expression was confirmed in patients. In brief, our research suggested that the SRPK1/GSK3ß axis promotes gefitinib resistance by activating the Wnt pathway and may serve as a potential therapeutic target for overcoming gefitinib resistance in NSCLC.

Gene expression study to elucidate the anti-trypanosomal activity of quinapyramine methyl sulphate (QPS).

The kinetoplastid protozoan parasite, Trypanosoma evansi causes a fatal disease condition known as Surra in equines throughout the globe. Disease condition being acute in nature, entrust a huge economic and health impact on the equine industry. Till date, quinapyramine methyl sulphate (QPS) is the first line of treatment and a panacea for the T. evansi infection in equines. Still after the >70 years of its discovery, there is no clue about the mode of action of QPS in T. evansi. The establishment of in vitro cultivation of T. evansi in HMI-9 media has provided opportunity to study the alteration in mRNA expression of parasite on exposure to the drug. With this research gap, the present study aimed to investigate the relative mRNA expression of 13 important drug target genes to elucidate the anti-trypanosomal activity of QPS against T. evansi. The IC50 of QPS against a pony isolate of T. evansi was determined as 276.4 nM(147.21 ng/ mL) in the growth inhibitory assay. The in vitro cultured T. evansi population were further exposed to IC50 of QPS and their relative mRNA expression was studied at 12 h, 24 h and 48 h interval.The mRNA expression of several genes such as hexokinase, trypanothione reductase, aurora kinase, oligopeptidase B and ribonucleotide reductase II were found refractory (non-significant, p > 0.1234) to the exposure of QPS. Significant up-regulation of trans-sialidase (p < 0.0001), ESAG8 (p < 0.0021), ribonucleotide reductase I (p < 0.0001), ornithine decarboxylase (p < 0.0001), topoisomerase II (p < 0.0021) and casein kinase I (p < 0.0021) were recorded after exposure with QPS. The arginine kinase 1 and calcium ATPase I showed highly significant (p < 0.0001) down-regulation in the drug kinetics. Therefore, the arginine kinase 1 and calcium ATPase I can be explored further to elucidate the trypanocidal activity of QPS. The preliminary data generated provide the potential of arginine kinase 1 and calcium ATPase I mRNA mediated pathway of trypanocidal action of QPS. Further, transcriptomics approach is required to investigate the possible mechanism of action of drugs at molecular level against the targeted organism.

Gene transfer of arginine kinase to skeletal muscle using adeno-associated virus.

In this study, we tested the feasibility of non-invasively measuring phosphoarginine (PArg) after gene delivery of arginine kinase (AK) using an adeno-associated virus (AAV) to murine hindlimbs. This was achieved by evaluating the time course, regional distribution and metabolic flux of PArg using (31)phosphorus magnetic resonance spectroscopy ((31)P-MRS). AK gene was injected into the gastrocnemius of the left hindlimb of C57Bl10 mice (age 5 weeks, male) using self-complementary AAV, type 2/8 with desmin promoter. Non-localized (31)P-MRS data were acquired over 9 months after injection using 11.1-T and 17.6-T Bruker Avance spectrometers. In addition, (31)P two-dimensional chemical shift imaging and saturation transfer experiments were performed to examine the spatial distribution and metabolic flux of PArg, respectively. PArg was evident in each injected mouse hindlimb after gene delivery, increased until 28 weeks, and remained elevated for at least 9 months (P<0.05). Furthermore, PArg was primarily localized to the injected posterior hindimb region and the metabolite was in exchange with ATP. Overall, the results show the viability of AAV gene transfer of AK gene to skeletal muscle, and provide support of PArg as a reporter that can be used to non-invasively monitor the transduction of genes for therapeutic interventions.