Toosendanin mediates cisplatin sensitization through targeting Annexin A4/ATP7A in non-small cell lung cancer cells.

Cisplatin (CDDP) is used in the treatment of non-small cell lung cancer (NSCLC), but due to the development of resistance, the benefit has been limited. Toosendanin (TSN) has shown therapeutic effects on NSCLC; however, the role of TSN on CDDP sensitization in NSCLC remains unknown. The antitumor effects of TSN and CDDP sensitization mediated by TSN were explored. TSN was added in various amounts to measure dose- and time-dependent cytotoxicity. Intracellular CDDP was detected by high-performance liquid chromatography. The protein levels of ATP7A, ATP7B, hCTR1, MRP-2, P-gp and Annexin A4 (Anxa4) were analyzed. The tests were conducted using normal NSCLC (A549 cell line) and CDDP-resistant cells (A549/DDP cell line). Anxa4 promotes CDDP resistance by regulating ATP7A, so Anxa4 was overexpressed and silenced and also transfected with pcMV6 or siRNA/ATP7A, respectively. Mechanistic investigations revealed that TSN decreased relative viability in NSCLC cells. Remarkably, TSN significantly enhanced CDDPsensitization in invalid doses. TSN downregulated Anxa4 expression, enhanced intracellular CDDP, and had no effect on MRP-2, P-gp, ATP7A, ATP7B or hCTR1. Subsequently, overexpression of Anxa4 led to a significant decrease in intracellular CDDP concentration. The adjustment of CDDP concentration regulated by TSN disappeared in Anxa4 or ATP7A-silenced cells. TSN also enhanced CDDP sensitization in single ATP7A-overexpressing cells, but had no effect on cells with simultaneous ATP7A overexpression and Anxa4 silencing. The present study suggests that TSN can mediate CDDP sensitization in NSCLC through downregulation of Anxa4.

Low-frequency stimulation of bilateral anterior nucleus of thalamus inhibits amygdale-kindled seizures in rats.

Brain stimulation with low-frequency is emerging as an alternative treatment for refractory epilepsy. The anterior nucleus thalamus (ANT) is thought to be a key structure in the circuits of seizure generation and propagation. The present study aimed to investigate the effects of low frequency stimulation (LFS) targeting ANT on amygdala-kindled seizures in Sprague-Dawley rats. Electrodes were implanted into the right basolateral amygdala and the right or bilateral ANT of Sprague-Dawley rats. When fully kindled seizures were achieved by daily electrical stimulation of the amygdala, LFS (15 min train of 0.1 ms pulses at 1 Hz and 200-500 µA) was applied to the unilateral or bilateral ANT immediately before the kindling stimulation (pre-treatment). Our study showed that LFS of the bilateral ANT significantly decreased the incidence of generalized seizures (GS) and seizure stage, as well as shortened duration of afterdischarge and GS demonstrating an inhibition of the severity of seizures. Moreover, LFS elevated the afterdischarge threshold (ADT) and GS threshold indicating an inhibition of susceptibility to seizures. On the other hand, LFS of the unilateral ANT failed to show any significance in inhibiting seizures. Our study demonstrated that bilateral LFS in ANT could significantly inhibit amygdala-kindled seizures by preventing both afterdischarge generation and propagation. It provided further evidence for clinical use of LFS in ANT.