Ying-Chih Huang

Background/Aim: Leukemia is a group of hematologic malignancies that present considerable therapeutic challenges worldwide. Isosakuranetin, a 4′-O-methylated flavonoid, possesses various biological properties. However, its therapeutic potential against leukemia remains unexplored because of an incomplete understanding of its molecular mechanisms. This study investigated the anticancer effects of isosakuranetin on leukemia cells and elucidated the associated signaling pathways. Materials and Methods: The human leukemia cell lines HL-60 and U937 were treated with serial concentrations of isosakuranetin. Cell viability was evaluated using the Cell Counting Kit-8 assay. Apoptosis and autophagy assays were conducted through annexin V/propidium iodide (PI) staining and acidic vesicular organelle (AVO) staining, respectively. Cell cycle distribution was analyzed through PI staining and flow cytometry. The expression of apoptosis- and autophagy-related proteins in leukemia cells was analyzed using western blotting. Results: Isosakuranetin exerted distinct regulatory effects on HL-60 and U937 cells over a 48-h treatment period. In HL-60 cells, it increased Beclin-1 expression and suppressed the JNK component of the mitogen-activated protein kinase pathway, up-regulated the PI3K/Akt signaling cascade, and modulated the expression of Bax and Bcl-2. Conversely, in U937 cells, isosakuranetin up-regulated both AMPK/PI3K/Akt and JNK signaling, while down-regulation of Beclin-1 expression. These pathway modulations collectively contributed to the induction of autophagy and apoptosis. Conclusion: Isosakuranetin induces apoptosis and autophagy in leukemia cells through the AMPK and PI3K/Akt pathways, as well as through JNK activation, with Beclin-1 playing a critical role in their crosstalk. These results highlight the potential of isosakuranetin as a therapeutic agent for leukemia.

Background/Aim: Chemotherapy drugs for leukemia, such as 5-azacytidine (Aza), have often various adverse effects. Hesperetin (Hes), a naturally occurring compound, is a potential adjuvant agent for anticancer therapy. This study aimed to investigate the effect of an Aza–Hes combination on acute leukemia cell lines, which elucidates the role of combination treatment in leukemia progression. Materials and Methods: HL-60 and U937 cells were treated with Aza and Hes at various concentrations or their combination. Cell proliferation and apoptosis was evaluated using the Cell Counting Kit-8 assay and annexin V/propidium iodide staining, respectively. Cell cycle analysis was conducted using flow cytometry. The expression of apoptosis-related and cell cycle–related proteins in leukemia cells was analyzed through western blotting. The synergistic effect of the Aza and Hes agents was estimated using the Chou–Talalay method. Results: We observed that Aza or Hes monotherapy engendered a dose-dependent reduction in HL-60 and U937 cell viability. However, treatment with the Aza–Hes combination for 24 h synergistically inhibited U937 cell proliferation by inducing both apoptosis and S-phase cell cycle arrest. Furthermore, the Aza–Hes combination down-regulated p-ERK and p-c-Jun N-terminal kinase expression and up-regulated p-p38 expression. Conclusion: Overall, our findings indicate that the Aza–Hes combination induces apoptosis and S-phase cell-cycle arrest through the mitogen-activated protein kinase pathway. In conclusion, the Aza–Hes combination is a potential antileukemia treatment.

Background: Hesperetin has been reported to have anticancer properties. However, the molecular mechanisms underlying its action on leukemia cells remain unclear. This in vitro study evaluated the possible mechanisms of hesperetin in leukemia cells (HL-60 and U937). Methods: Cell viability was evaluated using a cell counting kit-8 (CCK-8) assay. Apoptosis and autophagy assays were conducted through annexin V/PI staining and acidic vesicular organelle (AVO) staining. Cell cycle analysis was conducted through propidium iodide (PI) and flow cytometry. The expression of proteins related to apoptosis and autophagy, including cleaved-PARP-1, Bcl-2, Bax, LC3-I/II, Beclin-1, Atg5, p62, phospho-AMPK, AMPK, phospho-mTOR, mTOR, phospho-Akt, and Akt, in human leukemia cells were evaluated using Western blotting. Results: Hesperetin dose-dependently inhibited leukemia cell viability. However, we found a low degree of apoptosis and cell cycle arrest induced by hesperetin in U937 cells. These findings imply the presence of additional mechanisms modulating hesperetin-induced cell death. Next, we evaluated autophagy, the possible mechanism modulating cell death or survival, to clarify the underlying mechanism of hesperetin-induced cell death. Hesperetin also dose-dependently increased the ratio of LC3II/I, Atg5, and Beclin 1 and decreased p62. Moreover, 3-methyladenine (3-MA) and bafilomycin A1 (Baf-A1) inhibited hesperetin-induced autophagy. We suggest that hesperetin can protect cancer cells during the transient period and may extend survival. Furthermore, a decrease in p-mTOR and p-Akt expression and an increase in p-AMPK expression were observed. Collectively, these findings suggest that hesperetin induces autophagy by modulating the AMPK/Akt/mTOR pathway. Conclusion: Hesperetin promoted cell death in the human leukemic cell line U937 by inducing a low degree of slight apoptosis, cell cycle arrest, and autophagy. It is therefore a potential adjuvant to antileukemia therapy and may be combined with other chemotherapeutic drugs to reduce chemoresistance and side effects.

Very few studies have shown the real origin and timing of de novo variants (DNV) implicated in von Willebrand disease (VWD). We investigated four families with type 2 VWD. First, we conducted linkage analysis using single nucleotide variant genotyping to recognize the possible provenance of DNV. Second, we performed amplification refractory mutation system‐quantitative polymerase chain reaction to confirm the real origin of variant (~0% mutant cells) or presence of a genetic mosaic variant (0%–50% mutant cells) in three embryonic germ layer‐derived tissues and sperm cells. Then, three possible timings of DNV were categorized based on the relative likelihood of occurrence according to the number of cell divisions during embryogenesis. Two each with type 2B VWD (proband 1 p.Arg1308Cys, proband 4 p.Arg1306Trp) and type 2A VWD (proband 2 p.Leu1276Arg, proband 3 p.Ser1506Leu) were identified. Variant origins were identified for families 1, 2 and 3 and confirmed to originate from the mother, father and father, respectively. However, the father of family 4 was confirmed to have isolated germline mosaicism with 2.2% mutant sperm cells. Further investigation confirmed the paternal grandfather to be the origin of variant. Thus, we proposed that DNV originating from the two fathers most likely occurred at the single sperm cell, the one originating from the mother occurred at the zygote during the first few cellular divisions; alternatively, in family 4, the DNV most likely occurred at the early postzygotic development in the father. Our findings are essential for understanding genetic pathogenesis and providing accurate genetic counselling.

The low-density lipoprotein receptor (LDLR) gene has been reported to be associated with cerebral infarction. This study aimed to explore 2 genetic LDLR variants, rs688 and rs5925, for their potential roles in cerebral infarction. This genetic association study was conducted within an isolated Taiwanese population; 815 ischemic stroke patients (431 with atherothrombotic stroke and 384 with lacunar infarction) and 430 normal controls were enrolled. There was no significant difference in the genetic frequency of rs688 and rs5925 between the control group and overall ischemic stroke, atherothrombotic stroke, or lacunar infarct groups. However, when analyzing the association between the haplotypes related to rs688 and rs5925 and cerebral ischemic stroke, the most common haplotype allele CT was used as the reference allele, and the haplotype TC associated with a 65% increased risk of overall ischemic stroke, 72% increased risk of atherothrombotic stroke, and 70% increased risk of lacunar infarction; this indicated a synergistic effect between these 2 single-nucleotide polymorphisms. The LDLR analysis based on the haplotypes rs688 and rs5925 was conducted in a Taiwanese population and provided preliminary evidence suggesting that genetic polymorphisms of LDLR are associated with cerebral infarction.