Cofilin severs actin filaments and translocates to the nucleus. Though Ser3 dephosphorylation of cofilin is required for its actin-depolymerization activity, the mechanism regulating cofilin’s nuclear translocation remains unclear. In rat basophilic leukemia (RBL-2H3) cells, we previously reported that the transient Ser3 dephosphorylation in cofilin plays a role in actin polymerization/depolymerization after multivalent antigen stimulation. In this study, we revealed that multivalent antigen stimulation rapidly induced the nuclear localization of cofilin within 1 min. Ser3 dephosphorylation and cofilin’s nuclear localization were elicited by ionomycin addition, while pretreatment with BAPTA-AM inhibited the nuclear translocation of cofilin according to multivalent antigen stimulation, suggesting that the increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) is essential for the nuclear translocation of cofilin. Meanwhile, the dephosphorylation at Ser3 and nuclear localization of cofilin occurred in the absence of extracellular Ca²⁺ without multivalent antigen stimulation. Interestingly, the constitutively inactive form of cofilin (Ser3 to glutamic acid) was translocated to the nucleus following multivalent antigen stimulation, but not following extracellular Ca²⁺ depletion. These findings indicate that the increase in [Ca²⁺]_i induced by multivalent antigen triggers the nuclear translocation of cofilin independent of Ser3 dephosphorylation, while extracellular Ca²⁺ depletion-induced nuclear translocation of cofilin depends on Ser3 dephosphorylation. This suggests that distinct pathways regulate the nuclear translocation of cofilin based on Ca²⁺ dependency.

Antibody–drug conjugates (ADCs) have revolutionized targeted cancer therapy, with human epidermal growth factor receptor 2-targeted ADCs demonstrating notable clinical success. In contrast, the clinical efficacy of epidermal growth factor receptor (EGFR)-targeted ADCs has not yet been clearly demonstrated, owing to several underlying factors. In this study, we focused on one of the key determinants most directly linked to therapeutic efficacy, the intracellular uptake of ADCs, and examined the noncanonical endocytic pathway driven by p38-dependent phosphorylation of EGFR in PC-9 non-small cell lung cancer (NSCLC) cells harboring an EGFR exon 19 deletion. Using tumor necrosis factor-α to activate this pathway, we observed enhanced internalization of cetuximab–monomethyl auristatin E and increased cytotoxicity in vitro. Notably, we also found that cisplatin, the backbone of lung cancer chemotherapy, induces a similar noncanonical endocytic response, further supporting the physiological relevance of this pathway. Collectively, our results highlight noncanonical endocytosis as a tractable mechanism to enhance the intracellular delivery and antitumor activity of EGFR-targeted ADCs. This mechanistic insight provides a foundation for developing improved platforms and combination regimens capable of overcoming resistance in NSCLC.

Targeted α-radionuclide therapy (TAT) is a promising cancer therapy using α-emitters. However, therapeutic efficacy varies due to differences in the pharmacokinetics of TAT agents among patients. Vorinostat, a histone deacetylase inhibitor, is known to upregulate drug transporter expression, enhance the cellular uptake of β-emitting radiopharmaceuticals, and increase their antitumor efficacy. However, effective strategies or agents to enhance the tumor accumulation of TAT agents remain poorly understood. In this study, we focused on meta-[²¹¹At]At-astatobenzylguanidine ([²¹¹At]MABG) and investigated the potential utility of combining [²¹¹At]MABG with vorinostat. We evaluated protein expression of the norepinephrine transporter (NET), which is critical for the uptake of [²¹¹At]MABG, by immunoblotting in rat pheochromocytoma PC-12 cells and mouse embryonic fibroblast NIH/3T3 cells following vorinostat treatment. The synthesis of [²¹¹At]MABG was performed as previously described, and its cellular uptake was compared between vorinostat-treated and untreated cells. The cytotoxic effect was evaluated using MTT (3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) staining. NET expression in PC-12 cells increased from 24 h after vorinostat treatment, reaching approximately 2.8-fold at 48 h compared to control. No change in NET expression was observed in NIH/3T3 cells after vorinostat treatment. The cellular uptake of [²¹¹At]MABG in PC-12 cells increased by approximately 1.4-fold at 24 h post-vorinostat treatment. Moreover, colony growth of PC-12 cells after 24 h exposure to [²¹¹At]MABG was significantly reduced when combined with vorinostat. In conclusion, vorinostat increased NET expression and enhanced accumulation of [²¹¹At]MABG in pheochromocytoma. In addition, the combination of vorinostat and [²¹¹At]MABG significantly enhanced cytotoxicity. These results suggest that vorinostat is potentially beneficial in optimizing [²¹¹At]MABG therapy.

Peonidin is a compound derived from cyanidin, belonging to the anthocyanin group, which imparts a purplish-red color to flowers and fruits. It exhibits antioxidant, anti-inflammatory, and antiproliferative properties. This study investigated the anticancer properties of peonidin in human colon cancer cell lines. Normal and colon cancer cells were exposed to different doses of peonidin for different durations. Cell-based assays and Western blotting were performed to investigate the mechanisms driving peonidin activity. Peonidin exhibited cytotoxic effects on colon cancer cells in a dose- and time-dependent manner while sparing normal colon epithelial cells. Furthermore, peonidin triggered autophagy, as evidenced by the increase in autophagosomes in colon cancer cells. Western blot analysis revealed that peonidin elevated the levels of autophagy-related 4B, microtubule-associated protein 1 light chain 3-I/II, and sequestosome-1 (SQSTM/p62). Conversely, peonidin reduced cell invasion and the expression of matrix metalloproteinase-9, Snail, Slug, and vimentin. From a mechanistic standpoint, Western blotting indicated that peonidin might inhibit the extracellular signal-regulated kinase/nuclear factor-κB/cyclooxygenase-2 pathways, leading to autophagy induction and inhibition of cell invasion in colon cancer cells. These results provide preliminary evidence supporting the use of peonidin as a new potent candidate for colorectal cancer chemotherapy.

In 2024, we reported a brain-penetrant formulation of the vasoactive intestinal peptide receptor 2 (VIPR2) antagonist peptide KS-133 that mitigated cognitive dysfunction in the VIPR2 hyperactivation mouse model of schizophrenia. In this formulation, KS-133 was encapsulated in the hydrophobic core of nanoparticles (NPs) coated with 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine–KS-487 (DPPE–KS-487), a conjugate of the cyclic peptide KS-487 and DPPE produced by a click reaction that binds low-density lipoprotein-related protein 1, enabling blood–brain barrier penetration upon subcutaneous injection. However, the click reaction generated multiple positional isomers and manufacturing required repetitive cycles of ultrasonication at high and low temperatures, posing challenges for industrial scalability. In the current study, we coated KS-133-containing NPs with 1,2-distearoyl-sn-glycero-3-phosphoethanolamine–KS-487 (DSPE–KS-487), a novel conjugate of KS-487 with DSPE produced through a non-click method, thus eliminating positional isomers, and also established a simplified manufacturing process allowing a unidirectional transition from ultrasonication at high to low temperatures. This formulation remained physically and chemically stable for at least 12 months under refrigeration, with no changes in particle size, zeta potential, KS-133 content, or KS-487 presentation level. The formulation also exhibited brain penetration and therapeutic efficacy against VIPR2 agonist-induced novel object recognition impairment in mice comparable to those of DPPE–KS-487 NPs. Furthermore, no systemic side effects of hematologic, brain, heart, liver, and lung toxicity were detected following daily injections to mice for two weeks at five times the effective dose. This new KS-133 formulation incorporating DSPE–KS-487 as a brain-penetrant shuttle is a promising drug candidate for the treatment of cognitive dysfunction in schizophrenia.