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Background: The overproliferation of fibroblast-like synoviocytes (FLS) contributes to synovial hyperplasia, a pivotal pathological feature of rheumatoid arthritis (RA). Shikonin (SKN), the active compound from Lithospermum erythrorhizon, exerts anti-RA effects by diverse means. However, further research is needed to confirm SKN's in vitro and in vivo anti-proliferative functions and reveal the underlying specific molecular mechanisms., Purpose: This study revealed SKN's anti-proliferative effects by inducing both apoptosis and autophagic cell death in RA FLS and adjuvant-induced arthritis (AIA) rat synovium, with involvement of regulating the AMPK/mTOR/ULK-1 pathway., Methods: SKN's influences on RA FLS were assessed for proliferation, apoptosis, and autophagy with immunofluorescence staining (Ki67, LC3B, P62), EdU incorporation assay, staining assays of Hoechst, Annexin V-FITC/PI, and JC-1, transmission electron microscopy, mCherry-GFP-LC3B puncta assay, and western blot. In AIA rats, SKN's anti-arthritic effects were assessed, and its impacts on synovial proliferation, apoptosis, and autophagy were studied using Ki67 immunohistochemistry, TUNEL, and western blot. The involvement of AMPK/mTOR/ULK-1 pathway was examined via western blot., Results: SKN suppressed RA FLS proliferation with reduced cell viability and decreased Ki67-positive and EdU-positive cells. SKN promoted RA FLS apoptosis, as evidenced by apoptotic nuclear fragmentation, increased Annexin V-FITC/PI-stained cells, reduced mitochondrial potential, elevated Bax/Bcl-2 ratio, and increased cleaved-caspase 3 and cleaved-PARP protein levels. SKN also enhanced RA FLS autophagy, featuring increased LC3B, reduced P62, autophagosome formation, and activated autophagic flux. Autophagy inhibition by 3-MA attenuated SKN's anti-proliferative roles, implying that SKN-induced autophagy contributes to cell death. In vivo, SKN mitigated the severity of rat AIA while also reducing Ki67 expression, inducing apoptosis, and enhancing autophagy within AIA rat synovium. Mechanistically, SKN modulated the AMPK/mTOR/ULK-1 pathway in RA FLS and AIA rat synovium, as shown by elevated P-AMPK and P-ULK-1 expression and decreased P-mTOR expression. This regulation was supported by the reversal of SKN's in vitro and in vivo effects upon co-administration with the AMPK inhibitor compound C., Conclusion: SKN exerted in vitro and in vivo anti-proliferative properties by inducing apoptosis and autophagic cell death via modulating the AMPK/mTOR/ULK-1 pathway. Our study revealed novel molecular mechanisms underlying SKN's anti-RA effects., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier GmbH. All rights reserved.)

Wogonin is a plant monoflavonoid which has been reported to inhibit cell growth and/or induce apoptosis in various tumors. The present study examined the apoptosis-inducing activity and underlying mechanism of action of wogonin in A549 cells. The results showed that wogonin was a potent inhibitor of the viability of A549 cells. Apoptotic protein changes detected after exposure to wogonin included decreased XIAP and Mcl-1 expression, increased cleaved-PARP expression and increased release of AIF and cytochrome C. Western blot analysis showed that the activity of c-Myc/Skp2 and HDAC1/HDAC2 pathways, which play important roles in tumor progress, was decreased. Quantitative PCR identified increased levels of c-Myc mRNA and decreased levels of its protein. Protein levels of Fbw7α, GSK3β and Thr58-Myc, which are involved in c-Myc ubiquitin-dependent degradation, were also analyzed. After exposure to wogonin, Fbw7α and GSK3β expression decreased and Thr58-Myc expression increased. However, MG132 was unable to prevent c-Myc degradation. The present results suggest that wogonin has multiple anti-cancer effects associated with degradation of c-Myc, SKP2, HDAC1 and HDAC2. Its ability to induce apoptosis independently of Fbw7α suggests a possible use in drug-resistance cancer related to Fbw7 deficiency. Further studies are needed to determine which pathways are related to c-Myc and Fbw7α reversal and whether Thr58 phosphorylation of c-Myc is dependent on GSK3β.

Objective: To establish a hepatolenticular degeneration rat model with excessive copper, and investigate the effects of excessive copper deposits in the liver on hepatocyte apoptosis and Bax and Bcl-2 expression., Methods: Rat model of hepatolenticular degeneration was established by administering forages containing 1g/kg of copper sulfate and drinking water containing 0.185% copper sulfate. Copper level in the liver and serum and alanine aminotransferase (ALT) level in serum were measured using an atomic absorption spectrophotometer. The terminal deoxynucleotidyl transferase-mediated deoxyuridine 5-triphosphate nick-end labeling (TUNEL) method was used to detect hepatocyte apoptosis. Bax and Bcl-2 expression was observed by RT-PCR and imunohistochemistry staining. Quantification of positive cells was performed by image analyzer., Results: With more prolonged excessive copper ingestion, copper level in the liver and serum as well as ALT level in serum rose, and more apoptosis cells appeared in the liver. Bax and Bcl-2 expression increased significantly compared with controls fed a normal diet and progressively increased with more prolonged excessive copper ingestion. The progressively increased extent of Bcl-2 expression was lower than that of Bax expression, so the ratio of Bcl-2/Bax decreased with increasing excessive copper ingestion time., Conclusions: Excessive copper deposits in the liver can induce hepatocyte apoptosis through an up-regulation of Bax expression.

Background & Objective: Recent study found angiogenesis plays some important roles in tumor growth and metastasis. This study was to explore the inhibitory effect of angiogenesis inhibitor Suramin in combination with cisplatin (DDP) on the growth and lung metastasis of lung adenocarcinoma LA795 cell xenografts in mice., Methods: Highly metastatic LA795 cells were inoculated into the mammary fatty pad of 32 T739 mice to establish lung adenocarcinoma models. Four days after inoculation, the mice were randomized into 4 groups: the mice in control group received intraperitoneal injection of 0.2 ml normal saline everyday; the mice in DDP group received injection of DDP [2 mg. (kg.day)-1] at the 4th, 11th, and 18th days; the mice in Suramin group received injection of Suramin [10 mg. (kg.day)-1] everyday; the mice in combination group received injection of DDP [2 mg. (kg.day)-1] at the 4th, 11th, and 18th days, and Suramin [10 mg. (kg day)-1] everyday. All mice were killed 24 days later. Lung and subcutaneous tumors were examined histologically. The metastatic tumor foci on lung surface were observed, lung weight was measured, the occurrence rate of lung metastasis and the inhibitory rate of metastatic foci were calculated, subcutaneous tumor microvessel density (MVD), vascular endothelial growth factor (VEGF), and nuclear factor-kappaB (NF-kappaB) were determined by immunohistochemistry, and tumor cell apoptosis was measured by TUNEL method., Results: In DDP, Suramin, and combination groups, tumor growth was suppressed significantly, with growth inhibitory rates of 23.0%, 34.4%, and 56.3%, respectively (P<0.05). Necrosis and decrease of tumor vessels were observed in Suramin and combination groups. The expression of NF-kappaB was significantly lower and tumor cell apoptosis index was significantly higher in DDP, Suramin, and combination groups than in control group (P<0.01). The metastatic foci on lung surface were less in Suramin and combination groups than in DDP and control groups. The expression of MVD and VEGF in subcutaneous tumors and the occurrence rate of lung metastasis were also obviously lower in Suramin and combination groups., Conclusion: Suramin has synergetic inhibitory effect with DDP on growth and metastasis of lung adenocarcinoma LA795 cell xenografts in mice through inhibiting angiogenesis and inducing cell apoptosis.

Objective: To investigate the apoptosis-inducing effect of cantharidin in human lung cancer cells A549 and its molecular mechanisms., Methods: MTT assay was used to determine A549 cells proliferation. Light and electron microscopy, FACScan, Annexin V-FITC staining and DNA gel electrophoresis were used to detect apoptosis. The expression of bcl-2, Bax and survivin were examined by Western blot., Results: Cantharidin inhibited the proliferation of A549 cells. The cells treated with cantharidin showed a typical apoptotic morphology and hypodiploid peak before G(1) phase. Flow cytometry analysis with annexin quantitatively further confirmed the increase of cell apoptosis. DNA of treated A549 cells depicted a ladder pattern characteristic of apoptosis, indicating the presence of DNA fragmentation. Western blot assay showed that cantharidin increased the level of Bax expression and inhibited the level of bcl-2 and survivin expression., Conclusion: Cantharidin can induce A549 cells apoptosis mainly via regulation of Bax, bcl-2 and survivin expression.

Aim: To explore the effects of hypoxia on Caspases activation in cardiomyocyte and role of intracellular calcium in this event in cardiomyocytes., Methods: After hypoxia 0 min, 30 min, 1 h, 3 h, 6 h, 12 h, 24 h, apoptotic cell percentage was determined with Hoechst 33342 straining. Expressions of Caspases-3 mRNA and release of mitochondrial cytochrome c in primary culture of cardiomyocytes were determined by using RT-PCR and Western blotting respectively., Results: Elevation of Cyt c in cytosol was in accordance with the decline in mitochondrial Cyt c content. Significant increase in Cyt c in cytosol appeared at 12 h post hypoxia and peaked at 24 h while Cyt c in mitochondria could not be detected at 24 h post hypoxia. Hypoxia up-regulated Caspases-3 mRNA expressions beginning at 3 h post hypoxia. Intracellular calcium overload occurred earlier than release of mitochondrial Cyt c and the activation of Caspase-3 during the hypoxic insult. Inhibition of Caspase-3 activation and pretreatment with calcium chelator BAPTA/AM offered a marked protective effect on hypoxia induced cardiomyocyte apoptosis., Conclusion: Hypoxia can induce mitochondrion-dependent Caspase-3 activation in cardiomyocytes and therefore leads to cell apoptosis. Increase of intracellular Ca2+ plays an important role in the activation of Caspase-3 and the induction of apoptosis in cardiomyocytes.

Objective: To investigate the mechanisms underlying the effect of selenium dioxide (SeO(2)) on the proliferation, apoptosis, and apoptosis-related gene expressions of Bcl-2 and p53 in 3 leukemia cell lines NB4, K562 and HL-60., Methods: The three leukemia cell lines were treated with 3, 10 and 30 mmol/L SeO(2) and apoptosis detected by flow cytometry and analysis of p53 and Bcl-2 expressions., Results: SeO(2) at 10 and 30 mmol/L could inhibit the proliferation of three leukemia cell lines. SeO(2) treatment at 30 mmol/L for 48 h induced an apoptosis rate of 54.0 %, 46.5 %, 49.6 % in NB4, K562, and HL-60 cells respectively, and down-regulated Bcl-2 expression in NB4 and K562 but not in HL-60 cells., Conclusion: SeO(2) can induce apoptosis in NB4, K562 and HL-60 leukemia cells, involving the down-regulation of Bcl-2 and up-regulation of p53.