Efficacy and safety of anlotinib plus anti-PD-1 agents in patients with refractory advanced biliary tract cancers.

BACKGROUND: Anlotinib has demonstrated promising anti-tumor efficacy in various solid tumors. Additionally, there is evidence suggesting that immune therapy can enhance the systemic responses of anlotinib. This study aimed to assess the effectiveness and safety of combining anlotinib with PD-1 inhibitors compared to fluoropyrimidine-based chemotherapy as a second-line treatment option for advanced biliary tract cancers (BTCs). METHODS: A total of 242 patients with BTCs were screened at the First Affiliated Hospital of Zhengzhou University from October 2015 to October 2022. Among them, 78 patients who received either anlotinib plus PD-1 inhibitors (AP) or fluoropyrimidine-based chemotherapy (FB) as second-line treatment were included in the study. The primary endpoint was progression-free survival (PFS), and the secondary endpoints included objective response rate (ORR), disease control rate (DCR), overall survival (OS), safety, and predictive tumor biomarkers. RESULTS: Among the 78 patients with BTCs, 39 patients received AP, while 39 patients were administered FB. The ORR in the AP group was 20.5%, compared to 5.1% in the FB group. The DCR was 87.2% in the AP group and 66.7% in the FB group. The AP group demonstrated significantly better ORR and DCR compared to the FB group (p = 0.042, p = 0.032). The median PFS and OS in the AP group were 7.9 months (95% CI: 4.35-11.45) and 13.9 months (95% CI: 5.39-22.41), respectively. In the FB group, the median PFS and OS were 4.1 months (95% CI: 3.17-5.03) and 13.2 months (95% CI: 8.72-17.68), respectively. The AP group exhibited significantly better median PFS than the FB group (p = 0.027). In the subgroup analysis, patients without liver metastasis had a much longer PFS in the AP group compared to the FB group (14.3 vs. 5.5 months, p = 0.016). Similarly, patients with CEA ≤ 5 µg/L also demonstrated a longer PFS in the AP group compared to the FB group (8.7 vs. 3.9 months, p = 0.008). CONCLUSIONS: The combination of anlotinib and PD-1 inhibitors demonstrated a promising clinical effect compared to fluoropyrimidine-based chemotherapy in the second-line treatment of refractory advanced BTCs. Liver metastases and CEA levels may serve as predictive factors for identifying patients who may benefit from AP therapy.

DNMT1 regulates hypermethylation and silences hsa_circ_401351 in hydroquinone-induced malignant TK6 cells.

BACKGROUND: Benzene and its metabolite hydroquinone (HQ) are widely used in daily life, and long-term exposure to benzene or HQ can induce acute myeloid leukemia (AML). Circular RNAs (circRNAs) are mostly produced by reverse splicing of gene exon mRNA precursors. The modulation of circRNA expression is connected to leukemia progression; however, the molecular mechanism is still unknown. MATERIALS AND METHODS: In this study, the cells were divided into four groups: PBS control group (PBS-TK6), TK6 malignantly transformed cells induced by 10.0 µmol/L HQ (HQ-TK6), and HQ-TK6 cells treated with 5 µmol/L 5-AzaC (DNA methyltransferase inhibitor) for 24 h (HQ + 5-AzaC). HQ-TK6 cells were treated with 200 nmol/L TSA (histone deacetylation inhibitor) for 24 h (HQ + TSA). qRT-PCR was used to identify the differential hsa_circ_401351 expression between the four groups. We further determined the hsa_circ_401351 promoter methylation level with methylation-specific PCR. DNMT1 and DNMT3b were knocked down by CRISPR/Cas9 to elucidate the specific molecular mechanism of hsa_circ_401351 in HQ-TK6 cells. CCK-8 and flow cytometry detected cell proliferation and apoptosis, respectively, after hsa_circ_401351 was overexpressed in HQ-TK6 cells. RESULTS: Compared with the PBS-TK6 group, the expression of hsa_circ_401351 was found to be lower in the HQ-TK6 group. Nevertheless, treatment with 5-AzaC or TSA increased hsa_circ_401351 expression, with the upregulation being more pronounced in the TSA group. The expression of hsa_circ_401351 in the DNMT1 knockdown group was dramatically increased by 50% compared to that in the control group, and the DNA methylation level of the hsa_circ_401351 promoter region was decreased. When hsa_circ_401351 was overexpressed, HQ-TK6 cell proliferation was significantly slowed after 48 h compared with the control group. Flow cytometry showed that cells were mainly arrested in G1 phase, and apoptosis was significantly enhanced. Similarly, qRT-PCR and Western blot data showed significant reductions in Caspase-3 mRNA and protein production, and Bcl-2 mRNA levels were also elevated. CONCLUSIONS: Overall, our research showed that elevated DNMT1 expression in HQ-TK6 cells increased methylation levels and decreased expression of the hsa_circ_401351 promoter region, limiting its ability to suppress HQ-TK6 cell growth and enhance apoptosis.

Effects of temperature and charged vacancies on electronic and optical properties of ß-Ga2O3 after radiation damage.

ß-Ga2O3 as an ultra-wide bandgap material is widely used in space missions and nuclear reactor environments. It is well established that the physical properties of ß-Ga2O3 would be affected by radiation damage and temperature in such application scenarios. Defects are inevitably created in ß-Ga2O3 upon irradiation and their dynamic evolution is positively correlated with the thermal motion of atoms as temperature increases. This work utilizes first-principles calculations to investigate how temperature influences the electronic and optical properties of ß-Ga2O3 after radiation damage. It finds that the effect of p-type defects caused by Ga vacancies on optical absorption diminishes as temperature increases. The high temperature amplifies the effect of oxygen vacancies to ß-Ga2O3, however, making n-type defects more pronounced and accompanied by an increase in the absorption peak in the visible band. The self-compensation effect varies when ß-Ga2O3 contains both Ga vacancies and O vacancies at different temperatures. Moreover, in the case of Ga3- (O2+) vacancies, the main characters of p(n)-type defects caused by uncharged Ga0 (O0) vacancies disappear. This work aims to understand the evolution of physical properties of ß-Ga2O3 under irradiation especially at high temperatures, and help analyze the damage mechanism in ß-Ga2O3-based devices.

Polarized radiative transfer in seawater-in-oil emulsions floated on seawater considering the impact of oil absorption on seawater droplet scattering.

Among various remote sensing approaches, optical polarization remote sensing shows great advantages in identifying oil-water emulsions in seawater and has become one of the most promising detection technologies. Herein, we focus on exploring the sensitivity of polarized radiative transfer properties for oil emulsion polarization detection to the influence factors of viewing angle, droplet volume fraction and radius, incident wavelength, and emulsion thickness. The radiative properties of seawater droplets dispersed in crude oil are calculated using the improved Lorenz-Mie theory considering the absorption of crude oil as the host medium, after which the reflected Stokes vector and the degree of linear polarization (DOLP) of seawater-in-oil emulsions floating on seawater are obtained using the spectral element method. By analyzing the calculation results of a 0° viewing azimuth angle, the detection wavelength and viewing zenith angles corresponding to the highest sensitivity of the DOLP to the above factors are significantly different; thus, quantitative remote sensing detection of the droplet volume fraction, droplet diameter, and emulsion thickness is possible. Exploring the sensitivity of polarized remote sensing signals for oil emulsion polarization detection to the above factors is a prerequisite for quantitative polarization detection of oil emulsions.

PARP-1 inhibits DNMT1-mediated promoter methylation and promotes linc01132 expression in benzene-exposed workers and hydroquinone-induced malignant transformed cells.

Hydroquinone (HQ), one of the main active metabolites of benzene, can induce the abnormal expression of long non-coding RNA (lncRNA). Studies have shown that lncRNA plays an important role in the occurrence of hematologic tumors induced by benzene or HQ. However, the molecular mechanism remains to be elucidated. Here, we investigated the molecular mechanism by which poly(ADP-ribose)polymerase 1 (PARP-1) interacts with DNA methyltransferase 1 (DNMT1) to regulate promoter methylation mediated linc01132 expression in HQ-induced TK6 malignant transformed cells (HQ-MT). The results revealed that the expression of linc01132 was increased in benzene-exposed workers and HQ-MT cells. The methylation of linc01132 promoter region was inhibited. Furthermore, in HQ-MT cells treated with 5-Aza-2'-deoxycytidine (5-AzaC) (DNA methyltransferase inhibitor) or trichostatin A (TSA) (histone deacetylation inhibitor), the expression of linc01132 was increased due to the regulation of DNA promoter methylation level by inhibiting DNMT1 expression. The methylation level of linc01132 promoter was correlated negatively with the expression of linc01132 in benzene-exposed workers, indicating that DNA methylation may contribute the expression of linc01132. Knockout of DNMT1, not DNMT3b, increased the expression of linc01132 as well as the demethylation of linc01132 promoter in HQ-MT cells. It was found that by knockdown PARP-1, the expression of DNMT1 in the nucleus was increased by immunofluorescence confocal microscopy, leading to the inhibition of hypermethylation in the promoter region of linc01132. Therefore, PARP-1 inhibits DNA methyltransferase (DNMT)-mediated promoter methylation and plays a role in linc01132 expression in benzene-exposed workers or HQ-MT cells, and is associated with benzene or HQ induced leukemia progression.

Crosstalk of moderate ROS and PARP-1 contributes to sustainable proliferation of conditionally reprogrammed keratinocytes.

Conditionally reprogrammed cell (CRC) technique is a promising model for biomedical and toxicological research. In the present study, our data first demonstrated an increased level of PARP-1 in conditionally reprogrammed human foreskin keratinocytes (CR-HFKs). We then found that PARP inhibitor ABT-888 (ABT), reactive oxygen species (ROS) scavenger N-acetyl-l-cysteine (NAC), or combination (ABT + NAC) were able to inhibit cell proliferation, ROS, PARP-1, and ROS related protein, NRF2, and NOX1. Interestingly, knockdown of endogenous PARP-1 significantly inhibited cell proliferation, indicating that the increased PARP-1 expression was critical for CR. Importantly, we found that a moderate level of ROS contributed the cell proliferation and increased PARP-1 since knockdown of PARP-1 also inhibited the ROS. The similar inhibition of cell proliferation, ROS, and expression of PARP-1 and NRF2 proteins was observed when CR-HFKs were treated with hydroquinone (HQ), a key component from skin-lightening products. Moreover, the treatment of HQ plus treatment of ABT, NAC, or combination can further inhibit cell proliferation, ROS, expression of PARP-1, and NRF2 proteins. PARP-1 knockdown inhibited the population doubling (PDL) and treatment of HQ inhibited the PDL further, as well as the change of ROS. Finally, we discovered that pathways including cyclin D1, NRF2, Rb and pRb, CHK2, and p53, were involved in cell proliferation inhibition with HQ. Taken together, our findings demonstrated that crosstalk between ROS and PARP-1 involves in the cell proliferation in CR-HFKs, and that inhibition of CR-HFK proliferation with HQ is through modulating G1 cell cycle arrest.

Hsa_circ_0001944 regulates apoptosis by regulating the binding of PARP1 and HuR in leukemia and malignant transformed cells induced by hydroquinone.

Hydroquinone (HQ) is one of the major metabolites of benzene and can cause abnormal gene expression. It is a known carcinogen that alters cell cycle disruption and cell proliferation. However, its chemical mechanism remain a mystery. Circular RNAs (circRNAs) are a subtype of noncoding RNAs (ncRNAs) that play a variety of roles in biological processes. Hsa_circ_001944 expression was upregulated in 30 leukemia patients and HQ-induced malignant transformed TK6 cells. Hsa_circ_001944 silencing inhibited the growth of HQ-TK6 cells and halted the cell cycle. The silencing of hsa_circ_0001944 led to increased cell accumulation in G1 versus S phase, increased apoptosis in the sh1944 versus the shNC group, and increased levels of DNA damage (γ-H2AX), leading to cell cycle arrest. In summary, inhibition of hsa_circ_001944 restricted cell growth by inhibiting cell cycle arrest and induced growth of HQ-TK6 cells by modulating PARP1 expression. Hsa_circ_0001944 targeted HuR, which is a kind of RNA-binding protein, to control PARP1 expression via RNAinter, RBPmap, and RBPdb. Fluorescence in situ hybridization combined with immunofluorescent labeling and western blotting experiments showed that hsa_circ_001944 was able to dissociate HuR and PARP1 binding in HQ-TK6 cells, control PARP1 production, and ultimately alter the PARP1/H-Ras pathway.

Enhancer-associated regulatory network and gene signature based on transcriptome and methylation data to predict the survival of patients with lung adenocarcinoma.

Accumulating evidence has proved that aberrant methylation of enhancers plays regulatory roles in gene expression for various cancers including lung adenocarcinoma (LUAD). In this study, the transcriptome and methylation data of The Cancer Genome Atlas (TCGA)-LUAD cohort were comprehensively analyzed with a five-step Enhancer Linking by Methylation/Expression Relationships (ELMER) process. Step 1: 131,371 distal (2 kb upstream from the transcription start site) probes were obtained. Step 2: 10,665 distal hypomethylated probes were identified in an unsupervised mode with the get.diff.meth function. Step 3: 699 probe-gene pairs with negative correlations were screened using the get.pair function in an unsupervised mode. Step 4: After mapping with probes, 768 motifs were obtained and 24 of them were enriched. Step 5: 127 transcription factors (TFs) with differential expressions and negative correlations with methylation levels were screened, which were corresponding to 21 motifs. After the ELMER process, a prognostic "TFs-motifs-genes" regulatory network was constructed. The Least absolute shrinkage and selection operator (LASSO) and Stepwise regression analyses were further applied to identify variables in the TCGA-LUAD cohort and an eight-gene signature was constructed for calculating the risk score. The risk score was verified in two independent validation cohorts. The area under curve values of receiver operating characteristic curves predicting 1-, 3-, and 5-years survival ranged from 0.633 to 0.764. With the increase of the risk scores, both the survival statuses and clinical traits showed a worse tendency. There were significant differences in the degrees of immune cell infiltration, TMB values, and TIDE scores between the high-risk and low-risk groups. Finally, a better-performing prognostic nomogram was integrated with the risk score and other clinical traits. In short, this multi-omics analysis demonstrated the application of ELMER in analyzing enhancer-associated regulatory network in LUAD, which provided promising strategies for epigenetic therapy and prognostic biomarkers.

PARP-1 modulates the expression of miR-223 through histone acetylation to involve in the hydroquinone-induced carcinogenesis of TK6 cells.

The upstream regulators of microRNAs were rarely reported. Hydroquinone (HQ) is the main metabolite of benzene, one of the important environmental factors contributing to leukemia and lymphoma. In HQ-induced malignant transformed TK6 (TK6-HT) cells, the expression of PARP-1 and miR-223 were upregulated. When in PARP-1 silencing TK6-HT cells, miR-223 was downregulated and the apoptotic cell number correspondingly increased. In TK6 cells treated with HQ for different terms, the expression of miR-223 and PARP-1 were dynamically observed and found to be decreased and increased, respectively. Trichostatin A could increase the expression of miR-223, then the expression of HDAC1-2 and nuclear factor kappa B were found to be increased, but that of mH2A was decreased. PARP-1 silencing inhibited the protein expression of H3Ac, mH2A, and H3K27ac. By co-immunoprecipitation experiment, PARP-1 and HDAC2 were found to form a regulatory complex. In conclusion, we demonstrated that the upregulation of PARP-1 mediated activation of acetylation to promote the transcription of miR-223 possibly via coregulating with HDAC2, an epigenetic regulation mechanism involved in cell malignant transformation resulting from long-term exposure to HQ, in which course, H3K27ac might be a specific marker for the activation of histone H3, which also gives hints for benzene exposure research.

LINC00173 Interacts With DNMT1 to Regulate LINC00173 Expression via Promoter Methylation in Hydroquinone-Induced Malignantly Transformed TK6 Cells and Benzene-Exposed Workers.

Long-term exposure to benzene or its metabolite, hydroquinone (HQ), can causally contribute to acute myeloid leukemia. Long-noncoding RNAs are essential epigenetic regulators with critical roles in tumor initiation and malignant progression; however, the mechanism by which aberrantly expressed LINC00173 (long intergenic nonprotein coding RNA 173) regulates the pathogenesis of acute myeloid leukemia is not fully understood. Here, we found that the expression of LINC00173 decreased while the expression of DNA methyltransferase 1 (DNMT1) increased, and the methylation of LINC00173 promoter was negatively correlated with LINC00173 expression in GEPIA, CCLE databases, benzene-exposed workers, B-cell non-Hodgkin's lymphoma, K562, U937, or HQ-induced malignantly transformed TK6 (HQ-MT cells). Furthermore, in 5-aza-2'-deoxycytidine (DNA methyltransferase inhibitor) or trichostatin A (histone deacetylation inhibitor)-treated HQ-MT cells, the expression of LINC00173 was restored by reduced DNA promoter methylation levels. HQ-MT cells with DNMT1 knockout by CRISPR/Cas9 restored the expression of LINC00173 and inhibited the DNA methylation of its promoter as well as enrichment of DNMT1 to promoter. Overexpression of LINC00173 inhibited the expression of DNMT1, cell proliferation, tumor growth, enhanced chemosensitivity to cisplatin, and apoptosis in HQ-MT cells. LINC00173 interacts with DNMT1 to regulate the methylation of LINC00173 promoter. Overall, this study provides evidence that interaction between DNMT1 and LINC00173 regulates the expression of LINC00173 by regulating its promoter methylation level, thus regulating the function of HQ-MT cells in vitro and in vivo, providing a new therapeutic target for benzene-induced tumor.

Rotenone-Induced Neurodegeneration Is Enabled by a p38-Parkin-ROS Signaling Feedback Loop.

Rotenone, a component of pesticides, is widely used in agriculture and potentially causes Parkinson's disease (PD). However, the regulatory mechanisms of rotenone-induced PD are unclear. Here, we revealed a novel feedback mechanism of p38-Parkin-ROS regulating rotenone-induced PD. Rotenone treatment led to not only the activation of p38 but also Parkin inactivation and reactive oxygen species (ROS) overproduction in SN4741 cells. Meanwhile, p38 activation regulated Parkin phosphorylation at serine 131 to disrupt Parkin-mediated mitophagy. Notably, both p38 inhibition and Parkin overexpression decreased ROS levels. Additionally, the ROS inhibitor N-acetyl-l-cysteine (NAC) inhibited p38 and activated Parkin-mediated mitophagy. Both p38 inhibition and the ROS inhibitor NAC exerted a protective effect by restoring cell death and mitochondrial function in rotenone-induced PD models. Based on these results, the p38-Parkin-ROS signaling pathway is involved in neurodegeneration. This pathway represents a valuable treatment strategy for rotenone-induced PD, and our study provides basic research evidence for the safe use of rotenone in agriculture.

The DNMT1-associated lncRNA UCA1 was upregulated in TK6 cells transformed by long-term exposure to hydroquinone and benzene-exposed workers via DNA hypomethylation.

Exposure to benzene or its metabolite hydroquinone (HQ) is a risk factor for a series of myeloid malignancies, and long noncoding RNAs play an important role in the process of pathogenesis. Urothelial cancer-associated 1 (UCA1) functions as an oncogene in the development of acute myeloid leukemia. However, the association between DNMT1 and UCA1 with benzene or HQ exposure has not been explored. We characterized UCA1 expression in cells briefly exposed to HQ (HQ-ST cells) and HQ-induced malignantly transformed (TK6-HT cells) treated with 5-aza-2'-deoxycytidine (5-AzaC) or trichostatin A (TSA). Compared to that in control cells, UCA1 expression was increased, whereas DNMT1 was decreased in HQ-ST cells and TK6-HT cells treated with 5-AzaC or TSA. Moreover, UCA1 expression was also upregulated and positively correlated with benzene exposure time in benzene-exposed workers. Furthermore, the expression of UCA1 was negatively associated with the DNA methylation level of its promoter in benzene-exposed workers. DNMT1 rather than DNMT3b knockout in TK6-HT cells activated the expression of UCA1 by inducing its promoter hypomethylation. These results suggest that benzene or HQ exposure leads to UCA1 upregulation via DNA hypomethylation in the UCA1 promoter, which is mediated by DNMT1.

Effect of Megestrol Acetate Combined With Oral Nutrition Supplement in Malnourished Lung Cancer Patients: A Single-Center Prospective Cohort Study.

Background: The optimal treatment of cancer-related malnutrition remains unknown. A single-center prospective cohort study was performed to compare the efficacy of megestrol acetate (MA) combined with oral nutrition supplement (ONS) and MA alone for the treatment of lung cancer-related malnutrition. Methods: 76 eligible patients were prospectively enrolled in two arms, Arm 1 patients (n = 40, 52.6%) received MA 160 mg/d, and Arm 2 patients (n = 36, 47.4%) received MA 160 mg/d combined with ONS 55.8 g/t.i.d, all orally. All patients received anticancer therapy. Treatment duration was 3 months. The primary endpoints were improvements in body mass index (BMI) and Eastern Cooperative Oncology Group (ECOG) score. Secondary endpoints were assessed by appetite, mid-upper arm circumference (MAC), serum pre-albumin levels, and serum albumin levels. Results: Baseline levels were comparable between Arm 1 and Arm 2 patients. Compared with Arm 1, primary endpoints (BMI, P = 0.018; ECOG, P = 0.022) and secondary endpoints (MAC, P = 0.025; serum pre-albumin, P = 0.043; and serum albumin, P = 0.034) were improved significantly after treatment in Arm 2. While toxicity was negligible and comparable between Arm 1 and Arm 2. Conclusion: MA combined with ONS may be an effective and safe treatment option for lung cancer-related malnutrition patients. Clinical Trial Registration:www.clinicaltrials.gov, identifier ChiCTR2100049007.

Small extrachromosomal circular DNA (eccDNA): major functions in evolution and cancer.

Extrachromosomal circular DNA (eccDNA) refers to a type of circular DNA that originate from but are likely independent of chromosomes. Due to technological advancements, eccDNAs have recently emerged as multifunctional molecules with numerous characteristics. The unique topological structure and genetic characteristics of eccDNAs shed new light on the monitoring, early diagnosis, treatment, and prediction of cancer. EccDNAs are commonly observed in both normal and cancer cells and function via different mechanisms in the stress response to exogenous and endogenous stimuli, aging, and carcinogenesis and in drug resistance during cancer treatment. The structural diversity of eccDNAs contributes to the function and numerical diversity of eccDNAs and thereby endows eccDNAs with powerful roles in evolution and in cancer initiation and progression by driving genetic plasticity and heterogeneity from extrachromosomal sites, which has been an ignored function in evolution in recent decades. EccDNAs show great potential in cancer, and we summarize the features, biogenesis, evaluated functions, functional mechanisms, related methods, and clinical utility of eccDNAs with a focus on their role in evolution and cancer.

Triphenyl phosphate disturbs the lipidome and induces endoplasmic reticulum stress and apoptosis in JEG-3 cells.

Triphenyl phosphate (TPP) is a frequently used aryl organophosphate flame retardant. Epidemiological studies have shown that TPP and its metabolite diphenyl phosphate (DPP) can accumulate in the placenta, and positively correlated with abnormal birth outcomes. TPP can disturb placental hormone secretion through the peroxisome proliferator-activated receptor γ (PPARγ) pathway. However, the extent and mechanism of placental toxicity mediation by TPP remains unknown. In this study, we used JEG-3 cells to investigate the role of PPARγ-regulated lipid metabolism in TPP-mediated placental toxicity. The results of lipidomic analysis showed that TPP increased the production of triglycerides (TG), fatty acids (FAs), and phosphatidic acid (PA), but decreased the levels of phosphatidylethanol (PE), phosphatidylserine (PS), and sphingomyelin (SM). TG accumulation was accompanied by increased levels of sterol regulatory element binding transcription factor 1 (SREBP1), acetyl-coA carboxylase (ACC), and fatty acid transport protein (CD36). Although PPARγ and its target CCAAT/enhancer binding proteins (C/EBPα) was decreased, the TG content and gene expression of SREBP1, ACC, and CD36 decreased when TPP was co-exposed to the PPARγ antagonist GW9662. TPP also induced inflammatory responses, endoplasmic reticulum stress (ERS), and cell apoptosis. Expression of genes related to ERS and apoptosis were attenuated by GW9662. Together, these results show that TPP can disturb lipid metabolism and cause lipid accumulation through PPARγ, induce ERS, and cell apoptosis. Our findings reveal that the developmental toxicity of TPP through placental toxicity should not be ignored.

Regulatory loop between lncRNA FAS-AS1 and DNMT3b controls FAS expression in hydroquinone-treated TK6 cells and benzene-exposed workers.

Hydroquinone (HQ), one of the main metabolites of benzene, is a well-known human leukemogen. However, the specific mechanism of how benzene or HQ contributes to the development of leukemia is unknown. In a previous study, we demonstrated the upregulation of DNA methyltransferase (DNMT) expression in HQ-induced malignant transformed TK6 (HQ-TK6) cells. Here, we investigated whether a regulatory loop between the long noncoding RNA FAS-AS1 and DNMT3b exists in HQ-TK6 cells and benzene-exposed workers. We found that the expression of FAS-AS1 was downregulated in HQ-TK6 cells and workers exposed to benzene longer than 1.5 years via histone acetylation, and FAS-AS1 expression was negatively correlated with the time of benzene exposure. Restoration of FAS-AS1 in HQ-TK6 cells promoted apoptosis and inhibited tumorigenicity in female nude mice. Interestingly, treatment with a DNMT inhibitor (5-aza-2-deoxycytidine), histone deacetylase inhibitor (trichostatin A), or DNMT3b knockout led to increased FAS-AS1 through increased H3K27ac protein expression in HQ-TK6 cells, and DNMT3b knockout decreased H3K27ac and DNMT3b enrichment to the FAS-AS1 promoter region, which suggested that DNMT3b and/or histone acetylation involve FAS-AS1 expression. Importantly, restoration of FAS-AS1 resulted in reduced expression of DNMT3b and SIRT1 and increased expression of FAS in both HQ-TK6 cells and xenograft tissues. Moreover, the average DNMT3b expression in 17 paired workers exposed to benzene within 1.5 years was decreased, but that of the remaining 103 paired workers with longer exposure times was increased. Conversely, DNMT3b was negatively correlated with FAS-AS1 expression. Both FAS-AS1 and DNMT3b influenced the enrichment of H3K27ac in the FAS promoter region by regulating the expression of SIRT1, consequently upregulating FAS expression. Taken together, these observations demonstrate crosstalk between FAS-AS1 and DNMT3b via a mutual inhibition loop and indicate a new mechanism by which FAS-AS1 regulates the expression of FAS in benzene-related carcinogenesis.

Monomer gypenoside LI from Gynostemma pentaphyllum inhibits cell proliferation and upregulates expression of miR-128-3p in melanoma cells.

Gypenosides have anticancer activity against many cancers. Gypenoside LI is a gypenoside monomer from Gynostemma pentaphyllum, its pharmacological functions in melanoma have not been reported. In this study, we found that gypenoside LI had a potent cytotoxic effect on melanoma cells. Gypenoside LI can induce intrinsic apoptosis along with S phase arrest. Furthermore, gypenoside LI inhibited the colony formation ability of melanoma through inhibition of the Wnt/ß-catenin signaling pathway. Interestingly, we also found that gypenoside LI can induce the upregulation of the tumor suppressor miR-128-3p during melanoma apoptosis. In contrast, gypenoside LI induced apoptosis, cell cycle arrest, and inhibition of the Wnt/ß-catenin signaling pathway, which were abolished by overexpression of the miR-128-3p inhibitor in A375 cells. Taken together, these results showed that gypenoside LI could inhibit human melanoma cells through inducing apoptosis, arresting cell cycle at the S phase and suppressing the Wnt/ß-catenin signaling pathway in a miR-128-3p dependent manner.

Up-regulation of DNMT3b contributes to HOTAIRM1 silencing via DNA hypermethylation in cells transformed by long-term exposure to hydroquinone and workers exposed to benzene.

Benzene exposure is a risk factor of acute myeloid leukemia (AML), during such carcinogenesis long non-coding RNAs (lncRNAs) are important epigenetic regulators. HOTAIRM1 (HOXA transcript antisense RNA, myeloid-specific 1) plays an indispensable role in the development of AML. Hydroquinone (HQ) is one major metabolite of benzene and its ideal replacement in toxicology research. But the influence of benzene or HQ on HOTAIRM1 expression in AML associated pathway is still unclear. In the TK6 cells with short-term exposure to HQ (HQ-ST cells) or long term HQ exposure induced malignant transformed TK6 cells (HQ-MT cells), the relationship between DNMT3b and HOTAIRM1 was explored. Comparing to counterparts, HOTAIRM1 expression was increased firstly and then decreased in HQ-ST cells, and definitely decreased in HQ-MT cells; while the expression change tendency of DNMT3b was in contrast to that of HOTAIRM1. Moreover, the average HOTAIRM1 expression of 17 paired workers being exposed to benzene within 1.5 years was increased, but that of the remaining 92 paired workers with longer exposure time was decreased. Furthermore, in 5-AzaC (DNA methyltransferase inhibitor) or TSA (histone deacetylation inhibitor) treated HQ-MT cells, the expression of HOTAIRM1 was restored by reduced DNA promoter methylation levels. HQ-MT cells with DNMT3b knockout by CRISPR/Cas9 displayed the promoter hypomethylation and the increase of HOTAIRM1, also confirmed in benzene exposure workers. These suggest that long term exposure to HQ or benzene might induce the increase of DNMT3b expression and the promoter hypermethylation to silence the expression of HOTAIRM1, a possible tumor-suppressor in the AML associated carcinogenesis pathway.

Poly(ADP-ribose) polymerase-1 promotes expression of miR-155 by the up-regulation of methyl-CpG binding domain protein 2 in TK6 cells exposed to hydroquinone.

Hydroquinone (HQ), one of the major metabolites of benzene, can induce aberrant gene expression. MiR-155, a tumor activator, participates in various biological processes, including DNA damage response. However, the molecular mechanism of aberrant miR-155 expression is still not completely elucidated. Here, we investigated the mechanism of abnormal expression of miR-155 induced by poly(ADP-ribose)polymerase-1 (PARP-1) expression in HQ-treated TK6 lymphoblastoid cells. We examined the expression of genes related to abnormal expression of miR-155 to explore the reason for this phenomenon. The results of the present study showed that miR-155 was significantly increased and reactive oxygen species (ROS) were decreased in cells treated with HQ for 72 h compared with PBS-treated cells. Meanwhile, E4F1, PARP-1 and PARP-1 related co-regulators (NF-κB, HDAC1, and HDAC2), acetylated histone H3 (H3Ac) were increased in a concentration-dependent manner. Experiments for treatment with 5-AzaC (DNMTs inhibitor), TSA (HDACs inhibitor), DOX (to activate PARP-1) or MG132 (proteasome inhibitor) revealed that the MBDs and PARP-1 was positively associated with miR-155 expression. Moreover, in cells treated with HQ in conjunction with PARP-1 knockdown, expression of miR-155, H3Ac and MBD2 protein were decreased, compared with negative control. In conclusion, PARP-1 activates expression of miR-155 via acetylation by regulating MBD2 in TK6 cells exposed to HQ.