The metallothionein gene family codes for proteins involved in metal homeostasis and acute detoxification of non-essential toxic metal ions across the tree of life. We have previously documented increased cadmium (Cd) tolerance in field populations of the crustacean Gammarus fossarum exposed to chronic metallic contamination of geochemical origin. This tolerance is lost during maintenance of organisms in the laboratory, and is transmitted to offspring via parental effects. This study investigated whether the expression of the Cd-responsive metallothionein gene mt1 could be related to Cd-tolerance plasticity in G. fossarum. In eleven populations with different chronic Cd exposure history, we simultaneously assessed Cd-tolerance (mortality tests) and G. fossarum mt1 expression levels by RT-qPCR in the gills and caeca of adult males and in neonates. mt1 expression levels in the two organs were correlated to Cd-tolerance in field organisms and a loss of tolerance was observed in parallel with a decreased expression of mt1 in the caeca after maintenance in uncontaminated water. We also recorded a greater inducibility of mt1 expression in offspring of tolerant populations in the laboratory when re-exposed to Cd along with the bi-parental transmission of Cd-tolerance. These results suggest that the control of mt1 expression is involved in the plasticity of Cd-tolerance in gammarid populations with different histories of Cd exposure.

Carboxylesterases (CarEs) play a critical role in metabolic resistance to insecticides of insects. But fewer CarEs were associated with insecticide bioactivation in insects. Previous findings showed that four CarE genes were downregulated in the indoxacarb resistant populations of Spodoptera litura. In this study, qPCR verification showed that the expression of SlituCOE067 was downregulated in the resistant strains and gradually decreased after exposure to indoxacarb. Silencing of SlituCOE067 increased the cells viability of S. litura against indoxacarb, and further knockdown of SlituCOE067 reduced the sensitivity of larvae to indoxacarb. Overexpression of SlituCOE067 in transgenic fruit flies decreased the tolerance to indoxacarb. Molecular modeling and insecticide docking predicted that SlituCOE067 protein can bind tightly to indoxacarb instead of its activated product N-decarbomethoxylated metabolite (DCJW). Heterologous expression and metabolism experiment proved that recombinant SlituCOE067 can promote the activation of indoxacarb into DCJW, but cannot metabolize DCJW. These results comprehensively demonstrate that downregulation of SlituCOE067 can reduce the activation metabolism of indoxacarb and mediate the resistance of S. litura to indoxacarb. This study reveals a new mechanism of insecticide resistance caused by blocking the activation of insecticides in lepidoptera insects.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy associated with poor prognosis. Baicalein, a flavonoid extracted from the roots of Scutellaria baicalensis, traditionally used in Chinese medicine, has demonstrated potential in inhibiting cancer development and progression. However, its mechanism of action remains poorly understood, particularly regarding epigenetic gene regulation through m6A RNA methylation. In this study, three human PDAC cell lines and one nonmalignant cell line were employed. The effects of baicalein were examined using multiple assays, including RT-qPCR, MeRIP-qPCR, Western blotting, spheroid formation, RNA stability, and MTT, to evaluate cellular functions and m6A regulation. Baicalein significantly reduced cell viability, migration, invasion, and colony formation. It also downregulated FTO, an enzyme critical for m6A RNA demethylation. Knockdown of FTO replicated the effects of baicalein, underscoring its oncogenic role in PDAC. Bioinformatic analysis identified ZEB1-a key transcription factor in epithelial-to-mesenchymal transition-as an m6A-modified target regulated by FTO. Both baicalein treatment and FTO knockdown enhanced m6A modification and decreased ZEB1 mRNA stability, thereby suppressing stemness-related features. Rescue experiments further confirmed that baicalein disrupts the TGF-β/FTO/ZEB1 signaling axis, highlighting its therapeutic potential in PDAC. This study offers fundamental insights for the development of novel therapeutic strategies targeting PDAC.

Cadmium (Cd), a highly toxic heavy metal, severely inhibits plant growth. Salt alleviates Cd stress in halophytes, however, the molecular mechanisms governing salt-mediated regulation of Cd toxicity remain poorly understood. This study elucidates the protective mechanism of NaCl in Cd-stressed Suaeda salsa seedlings. Cd exposure suppressed seedling growth and induced membrane lipid peroxidation. Conversely, NaCl application not only maintained normal growth but also effectively ameliorated Cd-induced phytotoxicity, potentially through osmotic adjustment mechanisms. Notably, using ion flux analysis, we found that NaCl attenuated Cd2+ influx into root epidermal cells, thereby enhancing Cd resistance. Pharmacological inhibition studies confirmed that Na + competitively inhibits Cd2+ uptake through shared channels/transporters. Furthermore, RT-qPCR gene expression profiling revealed that NaCl coordinately activated both ionic compartmentalization and efflux systems through upregulating plasma membrane-localized SsSOS1 and tonoplast-associated SsNHX1 for Na + extrusion and vacuolar sequestration, enhancing Cd2+ compartmentalization via SsCAX and SsVHA-B mediated transport and maintaining cellular homeostasis through SsHKT1 and SsPIP-mediated regulation of water and K+ balance, or indirectly inhibit Cd2+ influx. It reveals that salt weakens Cd2+ influx and enhances Cd tolerance by activating a coordinated gene regulatory network in Suaeda salsa. This finding offers valuable insights into phytoremediation strategies for enhancing crop resilience in Cd-contaminated saline soils.

Cholera, a diarrheal disease caused by the gram-negative bacterium Vibrio cholerae, remains a global health threat in developing countries due to its high transmissibility and increased antibiotic resistance. There is a pressing need for alternative strategies, with an emphasis on anti-virulent approaches to alter the outcome of bacterial infections, given the increase in antimicrobial-resistant strains. V. cholerae causes cholera by secreting virulence factors in the intestinal epithelial cells. These virulence factors facilitate bacterial colonization and cholera toxin production during infection. Here, we demonstrate that sodium butyrate (SB), a small molecule, had no effect on bacterial viability but was effective in suppressing the virulence attributes of V. cholerae. The production of cholera toxin (CT) was significantly reduced in a standard V. cholerae El Tor strain and two clinical isolates when grown in the presence of SB. Analysis of mRNA and protein levels further revealed that SB reduced the expression of the ToxT-dependent virulence genes like tcpA and ctxAB. DNA-protein interaction assays, conducted at cellular (ChIP) and in vitro conditions (EMSA), indicated that SB weakens the binding between ToxT and its downstream promoter DNA, likely by blocking DNA binding. Furthermore, the anti-virulence efficacy of SB was confirmed in animal models. These findings suggest that SB could be developed as an anti-virulence agent against V. cholerae, serving as a potential alternative to conventional antibiotics or as an adjunctive therapy to combat cholera.

Histone deacetylases (HDACs) are a class of epigenetic regulators that play pivotal roles in key biological processes such as cell proliferation, differentiation, metabolism, and immune regulation. Based on this, HDAC inhibitors (HDACis), as novel epigenetic-targeted therapeutic agents, have demonstrated significant antitumor potential by inducing cell cycle arrest, activating apoptosis, and modulating the immune microenvironment. Current research is focused on developing highly selective HDAC isoform inhibitors and combination therapy strategies tailored to molecular subtypes, aiming to overcome off-target effects and resistance issues associated with traditional broad-spectrum inhibitors. This review systematically elaborates on the multidimensional regulatory networks of HDACs in tumor malignancy and assesses the clinical translation progress of next-generation HDACis and their prospects in precision medicine, providing a theoretical framework and strategic reference for the development of epigenetic-targeted antitumor drugs.

The widespread occurrence of microplastics (MP) and nanoplastics (NP) in the environment is commonly thought to negatively impact living organisms; however, there remains a considerable lack of understanding regarding the actual risks associated with exposure. Microorganisms, including pathogenic bacteria, frequently interact with MPs/NPs in various ecosystems, triggering physiological responses that warrant a deeper understanding. The present study experimentally demonstrated the impact of surface-functionalized differentially charged polystyrene (PS) NPs on the physiology of human pathogenic Escherichia coli O157:H7 and their influence on biofilm formation. Our results suggest that charged NPs can influence the growth, viability, virulence, physiological stress response, and biofilm lifestyle of the pathogen. Positively-charged NPs were found to have a bacteriostatic effect on planktonic cell growth and affect cellular viability and biofilm initiation compared to negatively charged and uncharged NPs. The transcriptomic and gene expression data indicated significant changes in the global gene expression profile of cells exposed to NPs, including the differential expression of genes encoding several metabolic pathways associated with stress response and virulence. Significant upregulation of Shiga-like toxin (stx1a), quorum sensing, and biofilm initiation genes was observed in NP-exposed biofilm samples. Overall, exposure to NPs did not significantly affect the survival of pathogens but affected their growth and biofilm development pattern, and most importantly, their virulence traits.

DET has a significant inhibitory activity against a range of cancer cells; however, its specific effects and underlying mechanisms in Non-Small Cell Lung Cancer (NSCLC) remain to be fully elucidated. This study aimed to investigate the potential mechanisms through which DET exerts its anti-neoplastic effects on NSCLC.

Antimicrobial peptides are small cationic molecules produced by eukaryotic cells to combat infection, as well as by bacteria for niche competition. Polymyxin B (PmB), a cyclic antimicrobial peptide, is used prophylactically in livestock and as a last-resort treatment for multidrug-resistant bacterial infections in humans. In this study, a transcriptomic analysis in Vibrio cholerae showed that expression of the uncharacterized gene ompV is stimulated in response to PmB. We found that ompV is organized in a conserved four-gene operon with the two-component system carRS and virK in V. cholerae. A virK deletion mutant and an ompV deletion mutant were more sensitive to antimicrobials, suggesting that both OmpV and VirK contribute to antimicrobial resistance. Our transcriptomic analysis showed that the efflux pump vexAB, a known effector of PmB resistance, was upregulated in an ompV-dependent manner in the presence of PmB. The predicted structure of OmpV revealed a lateral opening in the β-barrel wall with access to an electronegative pocket in the barrel lumen that can accommodate PmB. Such an interaction could facilitate intracellular signaling through a conformational change in OmpV. This provides the first evidence of a specialized operon governing multiple systems for antimicrobial resistance in V. cholerae.

Nuclear HER2 (N-HER2) predicts resistance to HER2-targeted therapy and poor prognosis of breast cancer patients, and the underlying mechanisms remain unclear. Here, we show that high expression of p21-activated kinase 5 (PAK5) is associated with HER2-targeted therapy resistance and poor outcomes of breast cancer patients. Excitingly, we find an increase in N-HER2 protein expression in patients with high PAK5 expression, who demonstrate resistance to trastuzumab treatment. PAK5 phosphorylates methyltransferase METTL14 on serine 399 to enhance m6A modification of lncRNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), leading to increased MALAT1 stability. The stabilized MALAT1 inhibits ubiquitin-proteasomal degradation of the N-HER2 by affecting the interaction of deubiquitinase USP8 and N-HER2, thereby promoting N-HER2 accumulation. Moreover, HER2 upregulates the expression of PAK5 and MALAT1, activating the HER2-MALAT1 positive feedback loop. Importantly, PAK5 promotes the therapeutic resistance of HER2-positive breast cancer cells by increasing N-HER2 protein both in vitro and vivo. These findings highlight PAK5 as a therapeutic target for combating trastuzumab resistance in HER2-positive breast cancer.

Aberrant methylation of the EphA7 promoter has been observed in cervical cancer (CC); however, its precise function and role in CC remain largely unknown. In this study, we investigated the role and molecular mechanisms of EphA7 promoter methylation in cervical carcinogenesis. First, our results indicated that the reactivation of EphA7 expression via a CRISPR demethylation tool (dCas9-Tet1) had antitumor effects. It restrained tumor proliferation and invasion while promoting apoptosis via the PI3K/AKT signaling pathway in both CaSki and SiHa cells. The upstream interacting factors were subsequently captured by CRISPR-mediated pull-down in situ, and the result revealed that SP1 and MAZ interacted with the promoter of EphA7. However, the perturbation results revealed that EphA7 expression was associated with SP1/DNMT1 but not MAZ. Furthermore, 17-β-estradiol (E2) can upregulate EphA7 expression through demethylation via the SP1/DNMT1 axis. A rescue experiment revealed that interference with SP1 expression could restore the effect of E2 on increasing the expression of EphA7 by upregulating estrogen receptor expression. In addition, EphA7 demethylation reduced the half-maximal inhibitory concentration (IC50) of cisplatin and paclitaxel. Pooled analysis revealed that EphA7 promoter hypermethylation was positively correlated with tumor purity but negatively correlated with immune cell infiltration, cytotoxic T lymphocyte (CTL) and immune checkpoint (IC) activity, and the expression of EphA7 was significantly positively correlated with tumor mutational burden (TMB), microsatellite instability (MSI) and the presence of single nucleotide variant (SNV) neoantigens, suggesting a better prognosis for patients with EphA7 promoter hypomethylation and high expression. Collectively, these findings indicate that targeted demethylation of the EphA7 promoter and restoration of endogenous EphA7 expression by dCas9-Tet1 are promising therapeutic approaches and are favorable for the prognosis of CC patients.

Objective. Endoplasmic reticulum (ER) stress and hypoxia are key factors for the effective growth of malignant tumors, including glioblastoma. The phosphoserine aminotransferase 1 (PSAT1) is an ER stress-responsive enzyme responsible for serine synthesis and necessary for tumor cell proliferation. The present study aims to investigate the regulation of the PSAT1 gene expression in U87MG glioblastoma cells and normal human astrocytes by ER stress and hypoxia depending on hydrocortisone, a native stress hormone used for co-treatment of glioblastoma and other malignant tumors. Methods. The U87MG glioblastoma cells and normal human astrocytes were used. Hypoxia was introduced by dimethyloxalylglycine. Tunicamycin was used for the induction of ER stress. Further, the cells were treated with hydrocortisone. RNA was extracted from cells after 4 h exposure to hydrocortisone, tunicamycin, and hypoxia. The expression level of the PSAT1 gene was studied by quantitative RT-PCR and normalized to ACTB mRNA. Results. We found that treatment of normal human astrocytes with hydrocortisone resulted in a decreased expression of the PSAT1 gene, but its expression in glioblastoma cells was resistant to this hormone action. However, hypoxia did not significantly change the expression of the PSAT1 gene in normal astrocytes, but strongly modified the effect of hydrocortisone on this gene expression. At the same time, hypoxia increased the expression of the PSAT1 gene in glioblastoma cells independently of hydrocortisone. Tunicamycin decreased the expression of this gene in normal astrocytes, but increased it in glioblastoma cells. In addition, the impact of tunicamycin on PSAT1 gene expression was suppressed by hypoxia in both normal astrocytes and glioblastoma cells and by hydrocortisone only in normal astrocytes. At the same time, the combined effect of hypoxia and hydrocortisone greatly enhanced the expression of the PSAT1 gene in tunicamycin-treated normal astrocytes and especially glioblastoma cells. Conclusion. The results of this study showed that hydrocortisone differentially controls the regulation of PSAT1 gene expression by ER stress and hypoxia in normal astrocytes and glioblastoma cells and that the combined effect of hydrocortisone and hypoxia greatly enhanced PSAT1 gene expression in tunicamycin-treated cells.

The pathophysiology of diabetes-induced brain injury involves pyroptosis, an inflammatory programmed cell death. This study aimed to investigate the potential protective effect of cranberry extract (CE) against diabetes-induced brain injury. Type 1 diabetes was induced by intraperitoneal injection of streptozotocin in rats. Brain tissue samples were investigated for biochemical determination of the reduced glutathione (GSH), superoxide dismutase (SOD), and malondialdehyde (MDA), and the quantitative RT-PCR for the gene expression of glial cell-derived neurotrophic factor (GDNF), lncRNA GAS-5, and pyroptosis markers. ELISA was used to determine the caspase-1 level and immunohistochemical staining for assessing IL-1β. Prophylactic dosing of the CE in diabetic rats improved cognitive behavior and significantly suppressed MDA concentration, pyroptosis genes expression (gasdermin D and caspase 1), and lncRNA GAS-5. In addition, CE significantly elevated GSH concentration, SOD activity, and gene expression of GDNF and markedly reduced IL-1β positive stained cells score in the brain. Phytochemical characterization of the CE by FT-IR and UPLC-PDA-MS/MS revealed cyanidin arabinoside, procyanidins, quercetin, and isorhamnetin as key components. CE protects against diabetes-induced cognitive dysfunction in rats by targeting redox-related signaling pathways and inducing an anti-inflammatory effect. LncRNA GAS-5 downregulation and pyroptosis pathway inhibition may contribute to its beneficial effects, suggesting its therapeutic potential.

Bupleurum chinense DC. a medicinal plant valued for saikosaponins (SSs) with antipyretic and hepatoprotective properties, faces constrained SS biosynthesis mediated by abscisic acid (ABA) during growth. Basic helix-loop-helix (bHLH) transcription factors (TFs) are hypothesized to participate in ABA signaling cascades, but their mechanistic role in SS regulation remains undefined. In this study, 20 differentially expressed BcbHLH genes were identified by transcriptomic profiling of ABA-induced hairy roots, with four MYC-family candidates (BcbHLH1-BcbHLH4) demonstrating ABA-responsive regulatory potential. ABA exposure (100 or 200 μmol/L, 24-72 h) induced dose-dependent SS reduction, while correlation analyses revealed coordinated expression between BcbHLH1-BcHMGR (r = 0.62) and BcbHLH4-BcBAS (r = 0.78), pinpointing these TFs as critical nodes in SS pathway modulation. Tissue-specific profiling showed predominant BcbHLH expression in stems and young leaves, with nuclear localization confirming their transcriptional regulatory organelles. BcbHLH3/4 exhibited transcriptional activation activity in the MYC_N domain, while molecular docking predicted 11th Arginine in the HLH domain as essential for G-box DNA binding. Collectively, our findings suggest that BcbHLH1-BcbHLH4 may serve as potential switches for fine-tuning ABA responsiveness in SS biosynthesis. Strategic manipulation of BcbHLH activity through genetic engineering approaches such as CRISPR-based editing or overexpression could alleviate ABA-mediated biosynthetic repression. Furthermore, precision engineering of the critical functional domain in BcbHLH could enhance promoter-binding activity to target genes and improve SS biosynthesis efficiency. These findings provide a reference framework for harnessing transcriptional regulators to optimize SS production in Bupleurum chinense DC.

The plant hormone jasmonic acid (JA) can increase artemisinin content in Artemisia annua L., but the mechanism regulating artemisinin biosynthesis needs further study. Basic helix-loop-helix (bHLH) transcription factors play important roles in plant growth and development, defense responses, secondary metabolism, etc. However, the role of bHLH transcription factors in response to JA signaling in artemisinin biosynthesis has not been well reported. Therefore, it is of great significance to investigate whether potential bHLH transcription factors in A. annua can regulate artemisinin biosynthesis through the JA signaling pathway. In this study, transcriptome data of A. annua treated with MeJA (Methyl Jasmonate) were used to identify candidate bHLH transcription factors that respond to JA. Furthermore, AabHLH5 and two potential JASMONATE-ZIM DOMAIN (JAZ) proteins interacting with AabHLH5 were identified through plant transformation, yeast two-hybrid and bimolecular fluorescence complementarity assays. Furthermore, the molecular mechanism by which bHLH5 participates in the JA signaling pathway and negatively regulates artemisinin biosynthesis was verified.

Insulin-like growth factor 1 (IGF-1) influences various aspects of embryogenesis, including embryonic development. This study investigated the effects of IGF-1 on early embryonic development in pig embryos, focusing on its interaction with the Wnt/β-catenin signaling pathway, a key regulator of cell adhesion and proliferation.

To explore the role of GFPT2 in the sensitivity of STK11/KRAS lung cancer cells to cisplatin chemotherapy, and its underlying mechanism.

RNA degradation systems (e.g., RNA decay and RNA interference) and the phytohormone abscisic acid (ABA) are both essential for plant growth, development, and adaptation to stress. Although the interplay between these pathways has been recognized, the molecular mechanisms governing their coordination remain poorly understood. In this study, we revealed that mutations in the 5'-3' RNA-degrading enzyme Ethylene Insensitive 5 (EIN5) result in hypersensitivity to ABA in Arabidopsis, whereas defects in the 3'-5' RNA turnover machinery (ski mutants) do not. The ABA hypersensitivity of ein5 mutants was mitigated by mutating components of the post-transcriptional gene silencing (PTGS) pathway, including DICER-LIKE 2 (DCL2)/DCL4, RNA-Dependent RNA Polymerase 1 (RDR1)/RDR6, and ARGONAUTE 1 (AGO1). ABA treatment substantially increased the abundance of coding-transcript-derived small interfering RNAs (ct-siRNAs) in ein5, predominantly from two genes, Nitrate Reductase 1 (NIA1) and NIA2. Further analysis suggested that NIA1 and NIA2 negatively regulate both the ABA biosynthesis and signaling pathways. The key transcription factor Abscisic Acid Insensitive 3 (ABI3) represses SKI3 expression by directly binding to its promoter, thereby promoting the production of NIA1/NIA2-derived ct-siRNAs, leading to the ABA hypersensitivity of ein5. Conversely, ABA enhances the accumulation of EIN5 as well as DCL4 and AGO1, pointing to distinct regulation of the mRNA decay and PTGS pathways. Collectively, these findings demonstrate the pivotal roles of NIA1 and NIA2 in plant responses to abiotic stress and provide new insights into the interplay between the ABA response and RNA degradation pathways.

The mycotoxin deoxynivalenol (DON) is of high importance among feed contaminants because of its frequent occurrence in toxicologically relevant concentrations worldwide. Cereal crops, the main component of chicken diet, are commonly contaminated with DON, resulting in frequent exposure of chickens to DON. Likewise, Campylobacter (C.), a pathogen of major public and animal health concern, is frequently found in chicken flocks and poses a threat to the One Health approach. Campylobacter colonizes the gastrointestinal (GI) tract of poultry with a high bacterial load in the caeca. However, the mechanism of C. jejuni colonization in chickens is still not understood albeit it is well known that C. jejuni resides primarily in the mucosal layer of the chicken intestine. Therefore, in the actual study we focused on the effect of exposure to DON and/or C. jejuni on expression profiles of intestinal mucins (MUC1, MUC2), β-defensins (Gallinacin (GAL) 10, 12), cytokines (Toll-like receptor 2 (TLR2), Interleukin (IL) 6, 8, Interferon-γ (IFN)-γ), inducible nitric oxide synthase 2 (iNOS2), as well as selected tight junction proteins (Claudin 5 (CLDN5), Occludin (OCLN), and zonula occludens-1 (ZO1) via RT-qPCR. For this, a total of 150 one-day-old Ross 308 broiler chickens were randomly allocated to six different groups (n = 25 with 5 replicates/group) and were fed for 5 weeks with either contaminated diets (5 or 10 mg DON/kg feed) or basal diets (control). Following oral infection of birds with C. jejuni NCTC 12744 at 14 days of age, several changes in gene expression patterns were demonstrated. A significant (P ≤ 0.05) downregulation of MUC2 mRNA expression was observed in birds fed DON5 and DON10 diet, as well as in birds co-exposed to DON5 and C. jejuni at 7 dpi. Furthermore, at 14 dpi, MUC2 mRNA expression was significantly (P ≤ 0.05) downregulated in birds fed DON (5 mg and 10 mg/kg diet) with and without C. jejuni and in birds infected solely with C. jejuni. The actual study also demonstrated that co-exposure of broiler chickens to DON and C. jejuni resulted in a decreased barrier function via downregulation of OCLD mRNA expression. In addition, Campylobacter infection induced an increased expression of the antimicrobial peptide GAL12 and the IL8 gene, indicating that C. jejuni can initiate an immune response in the chicken gut in a proinflammatory manner. Similarly, DON with and without C. jejuni induced upregulation of GAL10 and GAL12 mRNA expression at 7 dpi. Moreover, no change in iNOS2 mRNA expression was observed in both the jejunum and the cecum at either 7 dpi or 14 dpi, suggesting unchanged NO production during exposure/infection. In conclusion, we confirmed that DON contamination corresponding to the currently applicable EU guidance value of 5 mg DON/kg feed affects the intestinal gene expression profiles of broilers, mainly in a dose-independent manner. Furthermore, DON exposure interacted synergistically with C. jejuni challenge regarding mucins, innate immunity gene expression in either the jejunum or the cecum, suggesting immunomodulatory activity of both foodborne agents (DON and C. jejuni).

Polyamines are small aliphatic amines whose metabolic reprogramming is involved in the regulation of various plant cellular reactions. Our previous study showed that polyamines increased lignan production in Linum album; however, little is known about the underlying mechanisms. This study aimed to provide more details on how putrescine (Put) regulates lignan biosynthesis in L. album cell culture. Our results showed that Put leads to podophyllotoxin (PTOX) and 6-methoxy podophyllotoxin (6MPTOX) accumulation by increasing the expression levels of phenylalanine ammonia-lyase (PAL) and pinoresinol-lariciresinol reductase (PLR) genes, encoding lignan biosynthesis regulatory enzymes. Put also increased hydrogen peroxide (H2O2) content, while its level decreased in the presence of aminoguanidine (AG) and imidazole, inhibitors of diamine oxidase (DAO) and NADPH oxidase (NOX), respectively. Elevated levels of nitric oxide (NO) and cytosolic free Ca2+ caused by Put treatment were reduced after using inhibitors of nitrate reductase (NR) and nitric oxide synthesis-like (NOS-like) enzymes, as well as Ca2+ influx. Besides, pre-treatment of cells with AG, imidazole, ethylene glycol-bis (β-aminoethyl ether)-N, N,N',N'-tetraacetic acid (EGTA) (Ca2+ chelator), Nɷ-nitro-L-arginine methyl ester (L-NAME), and Sodium tungstate (TUN) (NO generation inhibitors) diminished PAL and PLR transcript levels and PTOX and 6MPTOX accumulation, indicating the involvement of H2O2, NO, and Ca2+ in regulating lignan biosynthesis in L. album cells. Put also stimulated salicylic acid (SA) accumulation, being sensitive to all inhibitors used. Overall, this study suggests that Put-induced H2O2 generation in combination with NO and Ca2+ signals can regulate PAL and PLR genes expression and lignan production, likely in a SA-dependent manner.