Role of the PARP1/NF-κB Pathway in DNA Damage and Apoptosis of TK6 Cells Induced by Hydroquinone.

Hydroquinone(HQ) is a widely used industrial raw material and is a topical lightening product found in over-the-counter products. However, inappropriate exposure to HQ can pose certain health hazards. This study aims to explore the mechanisms of DNA damage and cell apoptosis caused by HQ, with a focus on whether HQ activates the nuclear factor-κB (NF-κB) pathway to participate in this process and to investigate the correlation between the NF-κB pathway activation and poly(ADP-ribose) polymerase 1(PARP1). Through various experimental techniques, such as DNA damage detection, cell apoptosis assessment, cell survival rate analysis, immunofluorescence, and nuclear-cytoplasmic separation, the cytotoxic effects of HQ were verified, and the activation of the NF-κB pathway was observed. Simultaneously, the relationship between the NF-κB pathway and PARP1 was verified by shRNA interference experiments. The results showed that HQ could significantly activate the NF-κB pathway, leading to a decreased cell survival rate, increased DNA damage, and cell apoptosis. Inhibiting the NF-κB pathway could significantly reduce HQ-induced DNA damage and cell apoptosis and restore cell proliferation and survival rate. shRNA interference experiments further indicated that the activation of the NF-κB pathway was regulated by PARP1. This study confirmed the important role of the NF-κB pathway in HQ-induced DNA damage and cell apoptosis and revealed that the activation of the NF-κB pathway was mediated by PARP1. This research provides important clues for a deeper understanding of the toxic mechanism of HQ.

Prenatal triphenyl phosphate exposure impairs placentation and induces preeclampsia-like symptoms in mice.

Triphenyl phosphate (TPhP) is an organophosphate flame retardant that is widely used in many commercial products. The United States Environmental Protection Agency has listed TPhP as a priority compound that requires health risk assessment. We previously found that TPhP could accumulate in the placentae of mice and impair birth outcomes by activating peroxisome proliferator-activated receptor gamma (PPARγ) in the placental trophoblast. However, the underlying mechanism remains unknown. In this study, we used a mouse intrauterine exposure model and found that TPhP induced preeclampsia (PE)-like symptoms, including new on-set gestational hypertension and proteinuria. Immunofluorescence analysis showed that during placentation, PPARγ was mainly expressed in the labyrinth layer and decidua of the placenta. TPhP significantly decreased placental implantation depth and impeded uterine spiral artery remodeling by activating PPARγ. The results of the in vitro experiments confirmed that TPhP inhibited extravillous trophoblast (EVT) cell migration and invasion by activating PPARγ and inhibiting the PI3K-AKT signaling pathway. Overall, our data demonstrated that TPhP could activate PPARγ in EVT cells, inhibit cell migration and invasion, impede placental implantation and uterine spiral artery remodeling, then induce PE-like symptom and impair birth outcomes. Although the exposure doses used in this study was several orders of magnitude higher than human daily intake, our study highlights the placenta as a potential target organ of TPhP worthy of further research.

Linc01588 deletion inhibits the malignant biological characteristics of hydroquinone-induced leukemic cells via miR-9-5p/SIRT1.

Leukemia caused by environmental chemical pollutants has attracted great attention, the malignant leukemic transformation model of TK6 cells induced by hydroquinone (HQ) has been previously found in our team. However, the type of leukemia corresponding to this malignant transformed cell line model needs further study and interpretation. Furthermore, the molecular mechanism of malignant proliferation of leukemic cells induced by HQ remains unclear. This study is the first to reveal the expression of aberrant genes in leukemic cells of HQ-induced malignant transformation, which may correspond to chronic lymphocytic leukemia (CLL). The expression of Linc01588, a long non-coding RNA (lncRNA), was significantly up-regulated in CLL patients and leukemic cell line model which previously described. After gain-of-function assays and loss-of-function assays, feeble cell viability, severe apoptotic phenotype and the increased secretion of TNF-α were easily observed in malignant leukemic TK6 cells with Linc01588 deletion after HQ intervention. The tumors derived from malignant TK6 cells with Linc01588 deletion inoculated subcutaneously in nude mice were smaller than controls. In CLL and its cell line model, the expression of Linc01588 and miR-9-5p, miR-9-5p and SIRT1 were negative correlation respectively in CLL and cell line model, while the expression of Linc01588 and SIRT1 were positive correlation. The dual-luciferase reporter assay showed that Linc01588 & miR-9-5p, miR-9-5p & SIRT1 could bind directly, respectively. Furthermore, knockdown of miR-9-5p successfully rescued the severe apoptotic phenotype and the increased secretion of TNF-α caused by the Linc01588 deletion, the deletion of Linc01588 in human CLL cell line MEC-2 could also inhibit malignant biological characteristics, and the phenotype caused by the deletion of Linc01588 could also be rescued after overexpression of SIRT1. Moreover, the regulation of SIRT1 expression in HQ19 cells by Linc01588 and miR-9-5 P may be related to the Akt/NF-κB pathway. In brief, Linc01588 deletion inhibits the malignant biological characteristics of HQ-induced leukemic cells via miR-9-5p/SIRT1, and it is a novel and hopeful clue for the clinical targeted therapy of CLL.

Triphenyl phosphate interferes with the synthesis of steroid hormones through the PPARγ/CD36 pathway in human trophoblast JEG-3 cells.

Triphenyl phosphate (TPhP), a chemical commonly found in human placenta and breast milk, has been shown to disturb the endocrine system. Our previous study confirmed that TPhP could accumulate in the placenta and interference with placental lipid metabolism and steroid hormone synthesis, as well as induce endoplasmic reticulum (ER) stress through PPARγ in human placental trophoblast JEG-3 cells. However, the molecular mechanism underlying this disruption remains unknown. Our study aimed to identify the role of the PPARγ/CD36 pathway in TPhP-induced steroid hormone disruption. We found that TPhP increased lipid accumulation, total cholesterol, low- and high-density protein cholesterol, progesterone, estradiol, glucocorticoid, and aldosterone levels, and genes related to steroid hormones synthesis, including 3ßHSD1, 17ßHSD1, CYP11A, CYP19, and CYP21. These effects were largely blocked by co-exposure with either a PPARγ antagonist GW9662 or knockdown of CD36 using siRNA (siCD36). Furthermore, an ER stress inhibitor 4-PBA attenuated the effect of TPhP on progesterone and glucocorticoid levels, and siCD36 reduced ER stress-related protein levels induced by TPhP, including BiP, PERK, and CHOP. These findings suggest that ER stress may also play a role in the disruption of steroid hormone synthesis by TPhP. As our study has shed light on the PPARγ/CD36 pathway's involvement in the disturbance of steroid hormone biosynthesis by TPhP in the JEG-3 cells, further investigations of the potential impacts on the placental function and following birth outcome are warranted.

Human CYP1A1-activated aneugenicity of aflatoxin B1 in mammalian cells and its combined effect with benzo(a)pyrene.

Aflatoxin B1 (AFB1) is the most toxic mycotoxin and a proven human carcinogen that requires metabolic activation, known by cytochrome P450 (CYP) 1A2 and 3A4. Previous evidence showed that AFB1 is activated by human recombinant CYP1A1 expressed in budding yeast. Yet, the toxicity, in particular the genotoxicity of the reactive metabolites formed from AFB1 remains unclear. Humans could be exposed to both AFB1 and benzo(a)pyrene (BaP) simultaneously, thus we were interested in their combined genotoxic effects subsequent to metabolic activation by CYP1A1. In this study, molecular docking of AFB1 to human CYP1A1 indicated that AFB1 is valid as a substrate. In the incubations with AFB1 in human CYP1A1-expressed microsomes, AFM1 as a marking metabolite of AFB1 was detected. Moreover, AFB1 induced micronucleus formation in a Chinese hamster V79-derived cell line and in a human lung epithelial BEAS-2B cell line, both expressing recombinant human CYP1A1, V79-hCYP1A1 and 2B-hCYP1A1 cells, respectively. Immunofluorescence of centromere protein B stained micronuclei was dominant in AFB1-treated BEAS-2B cells exposed to AFB1, suggesting an aneugenic effect. Moreover, AFB1 elevated the levels of ROS, 8-OHdG, AFB1-DNA adduct, and DNA breaks in 2B-hCYP1A1 cells, compared with those in the parental BEAS-2B cells. Meanwhile, AFB1 increased CYP1A1, RAD51, and γ-H2AX protein levels in 2B-hCYP1A1 cells, which were attenuated by the CYP1A1 inhibitor bergamottin. Co-exposure of AFB1 with BaP increased 8-OHdG, RAD51, and γ-H2AX levels (indicating DNA damage). In conclusion, AFB1 could be activated by human CYP1A1 for potent aneugenicity, which may be further enhanced by co-exposure to BaP.

Exosomal derived miR-1246 from hydroquinone-transformed cells drives S phase accumulation arrest by targeting cyclin G2 in TK6 cells.

BACKGROUND: Hydroquinone (HQ), a major metabolite of benzene and known hematotoxic carcinogen. MicroRNA 1246 (miR-1246), an oncogene, regulates target genes in carcinogenesis including leukemia. This study investigates the impact of exosomal derived miR-1246 from HQ-transformed (HQ19) cells on cell-to-cell communication in recipient TK6 cells. METHODS: RNA sequencing was used to identify differentially expressed exosomal miRNAs in HQ19 cells and its phosphate buffered solution control cells (PBS19), which were then confirmed using qRT-PCR. The impact of exosomal miR-1246 derived from HQ-transformed cells on cell cycle distribution was investigated in recipient TK6 cells. RESULTS: RNA sequencing analysis revealed that 34 exosomal miRNAs were upregulated and 158 miRNAs were downregulated in HQ19 cells compared with PBS19 cells. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses predicted that their targets are enriched in cancer development-related pathways, such as MAPK signaling, microRNAs in cancer, apoptosis, PI3K-Akt signaling, cell cycle, Ras signaling, and Chronic myeloid leukemia. Eleven miRNAs were confirmed to have differential expression through qRT-PCR, with 6 upregulated (miR-140-3p, miR-551b-3p, miR-7-5p, miR-1290, miR-92a-3p, and miR-1246) and 5 downregulated (miR-183-5p, miR-26a-5p, miR-30c-5p, miR-205-5p, and miR-99b-3p). Among these, miR-1246 exhibited the highest expression level. HQ exposure resulted in a concentration-dependent increase in miR-1246 levels and decrease Cyclin G2 (CCNG2) levels in TK6 cells. Similarly, exosomes from HQ19 exhibited similar effects as HQ exposure. Dual luciferase reporter gene assays indicated that miR-1246 could band to CCNG2. After HQ exposure, exosomal miR-1246 induced cell cycle arrest at the S phase, elevating the expression of genes like pRb, E2F1, and Cyclin D1 associated with S phase checkpoint. However, silencing miR-1246 caused G2/M-phase arrest. CONCLUSION: HQ-transformed cells' exosomal miR-1246 targets CCNG2, regulating TK6 cell cycle arrest, highlighting its potential as a biomarker for HQ-induced malignant transformation.

miR-153-3p via PIK3R1 Is Involved in Cigarette Smoke-Induced Neurotoxicity in the Brain.

Cigarettes contain various chemicals that cause damage to nerve cells. Exposure to cigarette smoke (CS) causes insulin resistance (IR) in nerve cells. However, the mechanisms for a disorder in the cigarette-induced insulin signaling pathway and in neurotoxicity remain unclear. Therefore, we evaluated, by a series of pathology analyses and behavioral tests, the neurotoxic effects of chronic exposure to CS on C57BL/6 mice. Mice exposed to CS with more than 200 mg/m3 total particulate matter (TPM) exhibited memory deficits and cognitive impairment. Pathological staining of paraffin sections of mouse brain tissue revealed that CS-exposed mice had, in the brain, neuronal damage characterized by thinner pyramidal and granular cell layers and fewer neurons. Further, the exposure of SH-SY5Y cells to cigarette smoke extract (CSE) resulted in diminished insulin sensitivity and reduced glucose uptake in a dose-dependent fashion. The PI3K/GSK3 insulin signaling pathway is particularly relevant to neurotoxicity. microRNAs are involved in the PI3K/GSK3ß/p-Tau pathway, and we found that cigarette exposure activates miR-153-3p, decreases PI3K regulatory subunits PIK3R1, and induces Tau hyperphosphorylation. Exposure to an miR-153 inhibitor or to a PI3K inhibitor alleviated the reduced insulin sensitivity caused by CS. Therefore, our results indicate that miR-153-3p, via PIK3R1, causes insulin resistance in the brain, and is involved in CS-induced neurotoxicity.

MiR-7-5p targeted Rb regulating cell cycle is involved in hydroquinone-induced malignant progression in human lymphoblastoid TK6 cells.

MiR-7-5p has been demonstrated to inhibit tumorigenesis by limiting tumor cell proliferation, migration and invasion. However, its role in countering hydroquinone (HQ)-induced malignant phenotype of TK6 cells has remained unclear. The present study aimed to investigate whether miR-7-5p overexpression could restrain the malignant phenotype in TK6 cells exposed to HQ. The results displayed that HQ suppressed the expression of miR-7-5p and promoted cell cycle progression. Further investigations confirmed that miR-7-5p could decelerate the cell cycle progression by targeting Rb after acute HQ exposure. Through the regulation of the Rb/E2F1 signaling pathway, the overexpression of miR-7-5p mitigated HQ-induced malignant phenotype in TK6 cells by impeding cell cycle progression. In conclusion, miR-7-5p overexpression appears to be involved in HQ-induced malignant transformation by suppressing Rb/E2F1 signaling pathway, resulting in a deceleration of the cell cycle progression.

Triphenyl phosphate disrupts placental tryptophan metabolism by activating MAOA/ROS/NFκB.

Triphenyl phosphate (TPhP) is an organophosphate flame retardant widely distributed in the environment. The neurodevelopmental toxicity of TPhP has been observed in animals and humans. Previously, we found that prenatal TPhP exposure disturbed placental tryptophan metabolism, impaired neurodevelopment in male offspring, and induced abnormal neurobehavior; however, the underlying mechanisms are unknown. In this study, using the trophoblast cell line JEG-3, we found that TPhP altered gene and protein expression in the tryptophan metabolism pathway, inhibited the tryptophan-serotonin pathway, and activated the tryptophan-kynurenine pathway. Meanwhile, TPhP induced oxidative stress by activating monoamine oxidase A (MAOA), promoting inflammatory factors including nuclear factor kappa-B (NFκB), interleukin-6, and tumor necrosis factor α. The NFκB inhibitor sulfasalazine could alleviate the effects of TPhP on tryptophan metabolism disturbance. The MAOA inhibitor clorgyline or the antioxidant N-acetylcysteine can mitigate oxidative stress and eliminate TPhP-induced inflammatory factors and tryptophan metabolism disturbances. The data above suggest that TPhP disturbed tryptophan metabolism by activating NFκB through MAOA-mediated oxidative stress. Finally, using the mouse intrauterine exposure model, the results confirmed that TPhP induced oxidative stress, activated inflammatory factors, disturbed tryptophan metabolism, and increased the levels of the tryptophan metabolites serotonin, kynurenine, 3-hydroxykynurenine, and 3-hydroxyanthranilic acid in the placenta during the second trimester of pregnancy. Overall, TPhP can disturb placental tryptophan metabolism by activating the inflammatory factor NFκB, which was induced by MAOA-induced oxidative stress. The results of this study confirm that indirect exposure to xenobiotic compounds at an early life stage can impair offspring development and provide a novel perspective on the neurodevelopmental toxicity of TPhP.

[The Mechanisms of Piceatannol in Inhibiting the Malignant Biological Characteristics of Acute Myeloid Leukemia Cells].

OBJECTIVE: To explore the effect and molecular mechanism of Piceatannol on malignant biological characteristics of acute myeloid leukemia (AML) cells. METHODS: HL60, U937, HL60/ADR and U937/ADR cells were treated with different concentrations of Piceatannol. CCK-8 assay was used to detect cell proliferation. Cell apoptosis was detected by flow cytometry with Annexin V/PI double staining. The protein expressions of apoptosis, autophagy and related signaling pathways were detected by Western blot. Real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) was used to detect the expression changes of drug resistance genes in drug-resistant AML cell lines. RESULTS: The activity of HL60 and U937 cells could be inhibited by Piceatannol and induced apoptosis. When Piceatannol interfered with AML cells for 24 h, the ratio of autophagy marker LC3-II/LC3-I increased with the increase of concentration (r=0.672， r=0.549). When Piceatannol interfered with AML cells for 48 h, the expression of Bcl-2 protein was down-regulated and caspase-3 was hydrolyzed and activated. At the same time, the activation level of Akt/NF-κB signaling pathway was inhibited to induce programmed death of AML cells. Piceatannol can also down-regulate the expression of MRP1 and gradually weaken the chemotherapy resistance of AML drug-resistant cell lines, but it has a weak effect on the expression of BCRP and almost no effect on MDR1. CONCLUSION: Piceatannol can inhibit the proliferation of AML cells and induce programmed death, which may be related to the inhibition of Akt/NF-κB signaling pathway, the hydrolysis of caspase-3 and the down-regulation of Bcl-2 protein expression, and the suppression of the expression of some drug resistance genes.

Peptide aptamer-based time-resolved fluoroimmunoassay for CHIKV diagnosis.

BACKGROUND: Chikungunya virus (CHIKV) and Dengue virus (DENV) have similar clinical symptoms, which often induce misdiagnoses. Therefore, an antigen detection diagnostic system that can clearly identify these two viruses is desirable. METHODS: In this study, we developed a novel peptide with high affinity and specificity to CHIKV, and further constructed peptide aptamer-based TRFIA assay to efficiently detect CHIKV. Peptide aptamer B2 (ITPQSSTTEAEL) and B3 (DTQGSNWI) were obtained through computer-aided design and selected as CHIKV-specific peptide aptamers based on their high binding affinity, strong hydrogen bonding, and RMSD of molecular docking. Then, a sandwich-Time-Resolved Fluoroimmunoassay (TRFIA) was successfully constructed for the detection of the interaction between peptide aptamers and viruses. RESULTS: When using B2 as the detection element, highly specific detection of CHIKV E2 was achieved with detection limits of 8.5 ng/ml in PBS solution. Variation coefficient between inter-assay showed the disturbances received from the detection of clinical fluid specimens (including serum and urine), were also within acceptable limits. The detection limits for 10-fold dilution serum and urine were 57.8 ng/mL and 147.3 ng/mL, respectively. The fluorescent signal intensity exhibited a good linear correlation with E2 protein concentration in the range of 0-1000 ng/mL, indicating the potential for quantitative detection of E2 protein. CONCLUSIONS: These results demonstrate that the construction of peptide aptamers with high affinity and specificity provides an excellent method for rapid diagnostic element screening, and the developed peptide aptamer B2 contributed to better detection of CHIKV viral particles compared to traditional antibodies.

[Experimental Study on the Mechanism of Mangiferin Inhibiting Malignant Biological Characteristics of Multiple Myeloma and Exerting Anticancer Effect].

OBJECTIVE: To investigate the effect of pure Chinese herbal extract Mangiferin on the malignant biological behaviors of multiple myeloma (MM) cells, and to analyze the molecular mechanism of the anti-myeloma effect of Mangiferin, so as to provide experimental basis for MM replacement therapy. METHODS: U266 and RPMI8226 of human MM cell lines were intervened with different concentrations of Mangiferin. Cell proliferation was detected by CCK-8 method. Annexin V/PI double staining flow cytometry was used to detect cell apoptosis. Western blot was used to detect the expression of apoptosis and related signaling pathway proteins, and real-time quantitative polymerase chain reaction (qRT-PCR) was used to detect the expression of matrix metalloproteinase (MMP) and CXC chemokine receptor (CXCR) family. RESULTS: Mangiferin could inhibit the proliferation activity of U266 and RPMI8226 cells and induce cells apoptosis. After Mangiferin intervened in U266, RPMI8226 cells for 48 h, the expression of Bcl-2 family pro-apoptotic protein Bax was up-regulated, while the expression of survivin and Bcl-xL proteins was down-regulated and caspase-3 was hydrolyzed and activated to promote cell apoptosis, besides, the expression of Bcl-2 protein in U266 cells was also significantly down-regulated to induce apoptosis (P<0.05). After Mangiferin intervenes in MM cells, it can not only increase the expression level of tumor suppressor p53, but also induce programmed cell death of MM cells by inhibiting the expression of anti-apoptotic molecules and down-regulating the phosphorylation levels of AKT and NF-κB. In addition, after the intervention of Mangiferin, the expressions of CXCR4, MMP2 and MMP9 in U266 cells were down-regulated (P<0.05), while there is no effect on the expressions of CXCR2, CXCR7 and MMP13 (P>0.05). However, the expressions of CXCR4, MMP9, and MMP13 in RPMI8226 cells were down-regulated (P<0.01), the expression of MMP2 was weakly affected, and the expression of CXCR2 and CXCR7 was basically not affected (P>0.05). CONCLUSION: Mangiferin can inhibit the proliferation and induce apoptosis of MM cells, and its mechanism may be related to inhibiting the activation of NF-κB signaling pathway, affecting the expression of Bcl-2 family proteins, and inhibiting the expression of core members of MMP and CXCR family.

JNK1 activated pRb/E2F1 and inhibited p53/p21 signaling pathway is involved in hydroquinone-induced pathway malignant transformation of TK6 cells by accelerating the cell cycle progression.

Hydroquinone (HQ) is an important metabolites of benzene in the body, and it has been found to result in cellular DNA damage, mutation, cell cycle imbalance, and malignant transformation. The JNK1 signaling pathway plays an important role in DNA damage repair. In this study, we focused on whether the JNK1 signaling pathway is involved in the HQ-induced cell cycle abnormalities and the underlying mechanism. The results showed that HQ induced abnormal progression of the cell cycle and initiated the JNK1 signaling pathway. We further confirmed that JNK1 suppression decelerated the cell cycle progression through inhibiting pRb/E2F1 signaling pathway and triggering p53/p21 pathway. Therefore, we concluded that JNK1 might be involved in HQ-induced malignant transformation associated with activating pRb/E2F1 and inhibiting p53/p21 signaling pathway which resulting in accelerating the cell cycle progression.

Hydroquinone-induced endoplasmic reticulum stress affects TK6 cell autophagy and apoptosis via ATF6-mTOR.

Hydroquinone (HQ), one of the main active metabolites of benzene in vivo, 7is commonly used as a surrogate for benzene in in vitro studies and has been shown to be cytotoxic. The aim of this study was to investigate the role of endoplasmic reticulum stress (ERS) in HQ-induced autophagy and apoptosis in human lymphoblastoid cells (TK6) and how activating transcription factor 6 (ATF-6) is involved. We treated TK6 cells with HQ to establish a cytotoxicity model and found that HQ induced cellular ERS, autophagy and apoptosis by Western blot, flow cytometry and transmission electron microscopy. In addition, inhibition of both reactive oxygen species (ROS) and ERS inhibited cellular autophagy and apoptosis, suggesting that ERS may be induced by ROS, which in turn affects autophagy and apoptosis. Our study also found that HQ could inhibit ATF6 expression and mTOR activation. Knockdown of ATF6 enhanced autophagy and apoptosis levels and further inhibited mTOR activation; activation of ATF6 by AA147 enhanced cellular activity, suggesting that ATF6 may affect cellular autophagy and apoptosis through mTOR. In conclusion, our data suggest that ROS mediated ERS may promote autophagy and apoptosis by inhibiting ATF6-mTOR pathway after HQ treatment of TK6 cells.

[The Effects and Regulatory Mechanism of Targeting CXC Chemokine Receptor 1/2 Combined with Ara-C on the Malignant Biological Behaviors of U937 Cells of Acute Myeloid Leukemia].

OBJECTIVE: To investigate and analyze the effect of CXC chemokine receptor 1/2 (CXCR1/2) targeting inhibitor Reparixin combined with cytarabine (Ara-C) on the malignant biological behaviors of acute myeloid leukemia cells and its effect on the expression of the CXCR family, while exploring the accompanying molecular mechanism, providing scientific basis and reference for new molecular markers and targeted therapy for AML. METHODS: Acute myeloid leukemia U937 cells were treated with different concentrations of Reparixin, Ara-C alone or in combination, and the cell morphology was observed under an inverted microscope; Wright-Giemsa staining was used to detect cell morphological changes; CCK-8 method was used to detect cell proliferation; the ability of cell invasion was detected by Transwell chamber method; the ability of colony formation was detected by colony formation assay; cell apoptosis was detected by Hoechst 33258 fluorescent staining and Annexin V/PI double-staining flow cytometry; monodansylcadaverine(MDC) staining was used to detect cell autophagy; the expression of apoptosis, autophagy and related signaling pathway proteins was detected by Western blot and the expression changes of CXCR family were detected by real-time quantitative polymerase chain reaction (qRT-PCR). RESULTS: Reparixin could inhibit the proliferation, invasion, migration and clone formation ability of U937 cells. Compared with the single drug group, when U937 cells were intervened by Reparixin combined with Ara-C, the malignant biological behaviors such as proliferation, invasion and colony formation were significantly decreased, and the levels of apoptosis and autophagy were significantly increased (P<0.01). After Reparixin combined with Ara-C intervenes in U937 cells, it can up-regulate the expression of the pro-apoptotic protein Bax and significantly down-regulate the expression of the anti-apoptotic protein Bcl-2, and also hydrolyze and activate Caspase-3, thereby inducing cell apoptosis. Reparixin combined with Ara-C could up-regulate the expressions of LC3Ⅱ and Beclin-1 proteins in U937 cells, and the ratio of LC3Ⅱ/LC3Ⅰ in cells was significantly up-regulated compared with single drug or control group (P<0.01). MDC result showed that the green granules of vesicles increased significantly, and a large number of broken cells were seen (P<0.01). Reparixin combined with Ara-C can significantly inhibit the phosphorylation level of PI3K, AKT and NF-κB signaling molecule, inhibit the malignant biological behavior of cells by inhibiting the activation of PI3K/AKT/NF-κB pathway, and induce programmed cell death. Ara-C intervention in U937 cells had no effect on the expression of CXCR family (P>0.05). The expression of CXCR1, CXCR2, and CXCR4 mRNA could be down-regulated by Reparixin single-agent intervention in U937 cells (P<0.05), and the expression of CXCR2 was more significantly down-regulated than the control group and other CXCRs (P<0.01). When Reparixin and Ara-C intervened in combination, the down-regulated levels of CXCR1 and CXCR2 were more significant than those in the single-drug group (P<0.01), while the relative expressions of CXCR4 and CXCR7 mRNA had no significant difference compared with the single-drug group (P>0.05). CONCLUSION: Reparixin combined with Ara-C can synergistically inhibit the malignant biological behaviors of U937 cells such as proliferation, invasion, migration and clone formation, and induce autophagy and apoptosis. The mechanism may be related to affecting the proteins expression of Bcl-2 family and down-regulating the proteins expression of CXCR family, while inhibiting the PI3K/AKT/NF-κB signaling pathway.

Long non-coding RNA LINC01480 is activated by Foxo3a and promotes hydroquinone-induced TK6 cell apoptosis by inhibiting the PI3K/AKT pathway.

Long non-coding RNAs (lncRNAs) have been shown to play a critical role in the damage caused to the body by environmental exogenous chemicals; however, few studies have explored their effects during exposure to benzene and its metabolite, hydroquinone (HQ). An emerging lncRNA, LINC01480, was found to be associated with the immune microenvironment of some cancers, but its specific function remains unknown. Therefore, this study aimed to investigate the role of LINC01480 in HQ-induced apoptosis. The biological function of LINC01480 was investigated through gain-of-function and loss-of-function experiments. Mechanically, nuclear-cytoplasmic fractionation experiment, chromatin immunoprecipitation (ChIP), dual-luciferase reporter assay, and rescue experiments were performed. In this study, when TK6 cells were treated with HQ (0, 5, 10, and 20 µM) for 12, 24, 48, and 72 h, the expression of LINC01480 was increased in a dose-dependent manner. Meanwhile, the phosphorylation levels of PI3K and AKT decreased, and apoptosis increased. As compared to the control group, HQ-induced apoptosis was significantly reduced, and the relative survival rate of TK6 cells increased after silencing LINC01480, while overexpression of LINC01480 further sensitized TK6 cells to HQ-induced apoptotic cell death. LINC01480 negatively regulated the PI3K/AKT pathway in TK6 cells, and the apoptosis-inhibiting effect of LINC01480 silencing was reversed after inhibition of the PI3K/AKT pathway. In addition, ChIP and the dual-luciferase reporter assays showed that the transcription factor Foxo3a promoted LINC01480 transcription by directly binding to the promoter regions - 149 to - 138 of LINC01480. Moreover, short-term HQ exposure promoted the expression of Foxo3a. From these findings, we can conclude that LINC01480 is activated by Foxo3a, and promotes HQ-induced apoptosis by inhibiting the PI3K/AKT pathway, suggesting that LINC01480 might become a possible target for therapeutic intervention of HQ-induced toxicity.

Effect of mir-92a-3p on hydroquinone induced changes in human lymphoblastoid cell cycle and apoptosis.

Hydroquinone (HQ), one of the metabolites of benzene in humans, has significant hepatotoxic properties. Chronic exposure to HQ can lead to leukemia. In a previous study by this group, we constructed a model of malignant transformation of human lymphoblastoid cells (TK6) induced by chronic exposure to HQ with significant subcutaneous tumorigenic capacity in nude mice. miR-92a-3p is a tumor factor whose role in HQ-induced malignant transformation is not yet clear. In the present study, raw signal analysis and dual-luciferase reporter gene results suggested that miR-92a-3p could target and regulate TOB1, and the expression level of miR-92a-3p was significantly upregulated in the long-term HQ-induced TK6 malignant transformation model, while the anti-proliferative factor TOB1 was significantly downregulated. To investigate the mechanism behind this, we inhibited miR-92a-3p in a malignant transformation model and found a decrease in cell viability, a decrease in MMP-9 protein levels, a G2/M phase block in the cell cycle, and an upregulation of the expression of G2/M phase-related proteins cyclinB1 and CDK1. Inhibition of miR-92a-3p in combination with si-TOB1 restored cell viability, inhibited cyclin B1 and CDK1 protein levels, and attenuated the G2/M phase block. Taken together, miR-92a-3p reduced the cell proliferation rate of HQ19 and caused cell cycle arrest by targeting TOB1, which in turn contributed to the altered malignant phenotype of the cells. This study suggests that miR-92a-3p is likely to be a biomarker for long-term HQ-induced malignant transformation of TK6 and could be a potential therapeutic target for leukemia caused by long-term exposure to HQ.

Nrf2 affects hydroquinone-induces cell cycle arrest through the p16/pRb signaling pathway and antioxidant enzymes.

Hydroquinone (HQ), a well-known carcinogenic agent, induces oxidative stress, cell cycle arrest, apoptosis, and malignant transformation. As an antioxidant actor, the nuclear factor erythroid 2-related factor 2 (Nrf2) drives adaptive cellular protection in response to oxidative stress. The human lymphoblastoid cell line (TK6 cells) is widely used as a model for leukemia researches. In the present study, we focused on exploring whether Nrf2 regulatory cell cycle in TK6 cells upon HQ treatment and the underlying mechanisms. The results showed that the cell cycle arrest in TK6 cells induced by hydroquinone was accompanied by activation of the Nrf2 signaling pathway. We further clarified that Nrf2 loss accelerated cell cycle progression from G0/G1 to S and G2/M phases and promoted ROS production by downregulating the expression of SOD and GSH. Western blotting analysis indicated that Nrf2 regulated cell cycle progression via p16/pRb signaling pathways. Therefore, we conclude that Nrf2 is engaged in HQ-induced cell cycle arrest as well through p16/pRb and antioxidant enzymes.

Human CYP1B1 enzyme-mediated, AhR enhanced activation of aflatoxin B1 for its genotoxicity in human cells.

Aflatoxin B1 (AFB1) is a human procarcinogen known to be activated by cytochrome P450 (CYP) 1A2 and 3A4. In a previous study AFB1 caused chromosomal rearrangement in a yeast strain genetically engineered for stably expressing human CYP1B1. Yet, further verification of the effect of AFB1 in human cells, a potential role of the aryl hydrocarbon receptor (AhR), and CYP1B1-catalyzed AFB1 metabolism remain unidentified. In this study, a human hepatocyte (L-02) line and a human lymphoblastoid (TK6) cell line were genetically engineered for the expression of human CYP1B1, producing L-02-hCYP1B1 and TK6-hCYP1B1, respectively. They were exposed to AFB1 and analyzed for the formation of micronucleus and elevation of γ-H2AX (indicating double-strand DNA breaks); the metabolites formed by CYP1B1 from AFB1 after incubation of AFB1 with human CYP1B1 isoenzyme microsomes were determined by LC-MS. The results showed significantly more potent induction of micronucleus by AFB1 in L-02-hCYP1B1 and TK6-hCYP1B1 than in the parental (L-02 and TK6) cells, and the effects were reduced by (E)- 2,3',4,5'-tetramethoxystilbene, a specific CYP1B1 inhibitor. In the AFB1- CYP1B1 microsomes incubations AFM1, a known stable metabolite of AFB1, was detected. Moreover, in L-02 and TK6 cells, AFB1 apparently increased the protein levels of AhR, ANRT and CYP1B1, and caused the nuclear translocation of AhR and ARNT, the latter effect being blocked by BAY-218 (an inhibitor of AhR). In conclusion, this study indicates that human CYP1B1 is capable of metabolically activating AFB1 through the AhR signaling pathway.