CD74-ROS1 L2026M mutant enhances autophagy through the MEK/ERK pathway to promote invasion, metastasis and crizotinib resistance in non-small cell lung cancer cells.

The treatment of non-small cell lung cancer (NSCLC) patients harboring a proto-oncogene tyrosine-protein kinase c-ros oncogene 1 (ROS1) fusion gene has greatly benefited from the use of crizotinib. However, drug resistance inevitably occurs after 1 year of treatment. Clinical studies have shown that patients with an L2026M mutation in the ROS1 kinase domain account for about 6% of the total number of crizotinib-resistant cases, which is an important group that cannot be ignored. To explore the mechanism involved, we constructed the HLA class II histocompatibility antigen gamma chain (CD74)-ROS1 L2026M mutant gene by fusion polymerase chain reaction (PCR) and transfected it into H460 and A549 cells. We found that the invasion and metastasis abilities of drug-resistant cells were increased. The results of monodansylcadaverine (MDC) staining, Acridine orange (AO) staining, and western blot indicated that the autophagy level of CD74-ROS1 L2026M mutant NSCLC cells was increased compared with the CD74-ROS1 group, and the inhibition of autophagy could reverse the increased invasion and metastasis abilities caused by the L2026M mutation. In addition, the L2026M mutation led to excessive activation of the MEK/ERK pathway, and MEK inhibitors could reduce the autophagy level, invasion, and metastasis abilities of cells; additionally, this process could be blocked by rapamycin, an activator of autophagy. Furthermore, crizotinib treatment activated expression of Src homology region 2 domain-containing phosphatase-2 (SHP2; also known as PTPN11) to upregulate the MEK/ERK pathway, and the combination of MEK inhibitors and crizotinib increased apoptosis compared with crizotinib alone. In conclusion, our results indicate that the MEK/ERK pathway mediates the induction of invasion, metastasis, and crizotinib resistance through autophagy caused by CD74-ROS1 L2026M mutation in NSCLC cells, and targeting MEK could reverse these processes.

Targeting HMGA1 contributes to immunotherapy in aggressive breast cancer while suppressing EMT.

Metastasis is an obstacle to the clinical treatment of aggressive breast cancer (BC). Studies have shown that high mobility group A1 (HMGA1) is abnormally expressed in various cancers and mediates tumor proliferation and metastasis. Here, we provided more evidence that HMGA1 mediated epithelial to mesenchymal transition (EMT) through the Wnt/ß-catenin pathway in aggressive BC. More importantly, HMGA1 knockdown enhanced antitumor immunity and improved the response to immune checkpoint blockade (ICB) therapy by upregulating programmed cell death ligand 1 (PD-L1) expression. Simultaneously, we revealed a novel mechanism by which HMGA1 and PD-L1 were regulated by the PD-L1/HMGA1/Wnt/ß-catenin negative feedback loop in aggressive BC. Taken together, we believe that HMGA1 can serve as a target for the dual role of anti-metastasis and enhancing immunotherapeutic responses.

ZYY-B-2, a novel ALK inhibitor, overcomes resistance to ceritinib by inhibiting P-gp function and induces apoptosis through mitochondrial pathway in ceritinib-resistant H2228 cells.

Targeting the Echinoderm microtubule-associated protein-like 4 and anaplastic lymphoma kinase (EML4-ALK) fusion gene is a promising therapeutic strategy for non-small-cell lung cancer (NSCLC) patients. With the advent of the first- and second-generation ALK inhibitors, the mortality rate of lung cancer has shown a downward trend, but almost inevitably, patients will eventually develop resistance, which severely limits the clinical application. Hence, developing new ALK inhibitors which can overcome resistance is essential. Here, we synthesized a novel ALK inhibitor 1-[4-[[5-Chloro-4-[[2-[(1-methylethyl)sulfonyl]phenyl]amino]-2-pyrimidinyl]amino]-3-methoxyphenyl]-3-[2-(4-methyl-1-piperazinyl)-2-oxoethyl]-2-imidazolidinone (ZYY-B-2) based on the structure of the second-generation ALK inhibitor ceritinib. ZYY-B-2 exhibited impressive anti-proliferative effect in the EML4-ALK positive H2228 cells and ceritinib-resistant H2228 (H2228/Cer) cells. Meanwhile, ZYY-B-2 inhibited the activation of p-ALK in a concentration-dependent manner, and inactivated its downstream target proteins p-AKT and p-ERK to inhibit cell proliferation. Subsequently, we found that ZYY-B-2 blocked H2228 cells and H2228/Cer cells in G0/G1 phase and induced cells to undergo apoptosis through the mitochondrial pathway. The ability of its anti-proliferation and pro-apoptosis was significantly stronger than the second generation ALK inhibitor ceritinib. In addition, high expression of P-gp was found in H2228/Cer cells compared with H2228 cells. ZYY-B-2 could inhibit the expression of P-gp in a dose-dependent manner to overcome ceritinib resistance, and the suppression effect of ZYY-B-2 on P-gp might be related to its inhibition of PI3K/AKT signaling pathway. In summary, ZYY-B-2, a promising ALK inhibitor, shows potent activity against ceritinib-resistant cells, which provides experimental and theoretical basis for the further development of new ALK inhibitors.

Cadmium-resistant phosphate-solubilizing bacteria immobilized on phosphoric acid-ball milling modified biochar enhances soil cadmium passivation and phosphorus bioavailability.

Cadmium (Cd) can accumulate in agriculture soil from the regular application of phosphorus (P) fertilizer. Microbiological method is considered as a potentially effective strategy that can not only remediate the Cd-contaminated soil but also provide the phosphorus needed for crop growth. However, the toxicity of Cd may affect the activity of microorganisms. To solve this problem, Klebsiella variicola with excellent phosphate solubilization ability (155.30 mg L-1 at 48 h) and Cd adsorption rate (90.84 % with 10 mg L-1 Cd initial concentration) was firstly isolated and identified in this study. Then, a phosphoric acid and ball milling co-modified biochar (PBC) was selected as the carrier to promote the activities of K. variicola under Cd pollution. Surface characterization revealed that the promotion of K. variicola by PBC was mainly attributed to the large specific surface area and diverse functional groups. Compared to contaminated soil, microbial PBC (MPBC) significantly increased the pakchoi biomass and phosphorus (P) content, while the Cd content in leave and root of pakchoi (Brassica chinensis L.) decreased by 25.90-43.46 % (P < 0.05). The combined application also favored the transformation of the resistant P fractions to bioavailable P, and facilitated the immobilization of 20.12 % exchangeable Cd to reducible, oxidizable, and residual Cd in the treated soil. High-throughput sequencing revealed that the response of the soil microbial community to the MPBC was more beneficial than K. variicola or PBC alone. Therefore, the application of MPBC has the potential to act as an efficient, stable, and environmentally friendly sustainable product for Cd remediation and enhanced P bioavailability in agricultural production.

Construction of crizotinib resistant models with CD74-ROS1 D2033N and CD74-ROS1 S1986F point mutations to explore resistance mechanism and treatment strategy.

Targeted therapy is an essential treatment for non-small cell lung cancer (NSCLC) that is always associated with the drug resistance. c-ros oncogene 1 (ROS1) gene point mutation is one of the leading factors causing drug resistance. However, the point mutation cell models of crizotinib are challenging to obtain, causing few reports on the drug resistance mechanism and the treatment strategy. We constructed CD74-ROS1 D2033N and CD74-ROS1 S1986F point mutant plasmids by fusion PCR technology and transfected them into A549 cells. Western blot and MTT assay proved that the drug-resistant cell lines were successfully transfected. The transwell assay confirmed that the mutant cells' motor abilities were significantly increased compared with the wild-type group. In addition, focal adhesion kinase (FAK) was significantly increased in mutant cells. Moreover, crizotinib resistance occurred in the mutant cells through the activation of FAK / phosphatidylinositol 3-kinase (PI3K) / protein kinase B (AKT) pathway. Next, crizotinib was combined with defactinib, a FAK inhibitor, to further explore its therapeutic effect. The results showed that the combination could significantly inhibit the proliferation, invasion and migration of mutant cells. In conclusion, we proved that CD74-ROS1 D2033N and CD74-ROS1 S1986F point mutant NSCLC cells were resistant to crizotinib through the activation of FAK/PI3K/AKT signaling pathway, and inhibiting FAK/PI3K/AKT signaling pathway activation by defactinib could overcome drug resistance in mutant cells.

FAT4 activation inhibits epithelial-mesenchymal transition (EMT) by promoting autophagy in H2228/Cer cells.

As a tumor suppressor in lung cancer, FAT atypical cadherin 4 (FAT4) has a critical role in epithelial-mesenchymal transition (EMT). However, the role of FAT4 in ceritinib-resistant anaplastic lymphoma kinase (ALK) positive non-small cell lung cancer (NSCLC) EMT has not been reported. It is necessary to discuss the role of FAT4 in this process and its potential mechanism of interaction. We found that the expression level of FAT4 was downregulated markedly in ceritinib-resistant NCI-H2228 (H2228/Cer) cells. Jujuboside A, a FAT4 activator, diminished EMT and metastasis of H2228/Cer cells. Importantly, autophagy inhibition inverted the inhibitory effect of FAT4 activation on EMT. Furthermore, we found the regulatory action of FAT4 on autophagy was related to proteasome 26S subunit ubiquitin receptor and non-ATPase 4 (PSMD4) and proteasome 20S subunit beta 4 (PSMB4), and the inhibitory effect of autophagy on EMT might be related to ROS/NF-κB/IκB-α and Wnt/ß-catenin pathways. In conclusion, FAT4 activation can inhibit the process of EMT in H2228/Cer cells by promoting autophagy, which provides a potential target for ceritinib-resistant ALK positive NSCLC therapy.

1-(4-((5-chloro-4-((2-(isopropylsulfonyl)phenyl)amino)pyrimidin-2-yl)amino)-3-methoxyphenyl)-3-(2-(dimethylamino)ethyl)imidazolidin-2-one (ZX-42) inhibits cell proliferation and induces apoptosis via inhibiting ALK and its downstream pathways in Karpas299 cells.

Anaplastic lymphoma kinase (ALK) belongs to the family of receptor tyrosine kinases. Recently, the incidence of anaplastic large cell lymphoma (ALCL) with ALK rearrangement has raised considerably. The application of ALK-targeted inhibitors such as ceritinib provides an effective therapy for the treatment of ALK-positive cancers. However, with the prolongation of treatment time, the emergence of resistance is inevitable. We found that 1-(4-((5-chloro-4-((2-(isopropylsulfonyl)phenyl)amino)pyrimidin-2-yl)amino)-3-methoxyphenyl)-3-(2-(dimethylamino)ethyl)imidazolidin-2-one (ZX-42), a novel ceritinib derivative, could inhibit the proliferation of ALK-positive ALCL cells, induce the apoptosis of Karpas299 cells through the mitochondrial pathway in a caspase-dependent manner. In addition, ZX-42 could suppress ALK and downstream pathways including PI3K/Akt, Erk and JAK3/STAT3 and reduce the nuclear translocation of NFκB by inhibiting TRAF2/IKK/IκB pathway. Taken together, our findings indicate that ZX-42 shows more effective activity than ceritinib against ALK-positive ALCL. We hope this study can provide a direction for the structural modification of ceritinib and lay the foundation for the further development of clinical research in ALK-positive ALCL.

A novel tubulin inhibitor, 6h, suppresses tumor-associated angiogenesis and shows potent antitumor activity against non-small cell lung cancers.

Tumor angiogenesis is closely associated with the metastasis and progression of non-small cell lung cancer (NSCLC), a highly vascularized solid tumor. However, novel therapeutics are lacking for the treatment of this cancer. Here, we developed a series of 2-aryl-4-(3,4,5-trimethoxy-benzoyl)-5-substituted-1,2,3-triazol analogs (6a-6x) as tubulin colchicine-binding site inhibitors, aiming to find a novel promising drug candidate for NSCLC treatment. We first identified 2-(2-fluorophenyl)-3-(3,4,5-trimethoxybenzoyl)-5-(3-hydroxyazetidin-1-yl)-2H-1,2,3-triazole (6h) as a hit compound, which inhibited angiogenesis induced by NSCLC cells both in vivo and in vitro. In addition, our data showed that 6h could tightly bind to the colchicine-binding site of tubulin and inhibit tubulin polymerization. We also found that 6h could effectively induce G2/M cell cycle arrest of A549 and H460 cells, inhibit cell proliferation, and induce apoptosis. Furthermore, we showed 6h had the potential to inhibit the migration and invasion of NSCLC cells, two basic characteristics of tumor metastasis. Finally, we found 6h could effectively inhibit tumor progression in A549 xenograft mouse models with minimal toxicity. Taken together, these findings provide strong evidence for the development of 6h as a promising microtubule colchicine-binding site inhibitor for NSCLC treatment.

2-Methoxy-5((3,4,5-trimethosyphenyl) seleninyl) phenol causes G2/M cell cycle arrest and apoptosis in NSCLC cells through mitochondrial apoptotic pathway and MDM2 inhibition.

Nonsmall cell lung cancer (NSCLC) is one of the most common malignancies and needs novel and effective chemotherapy. In this study, our purpose is to explore the anticancer effects of 2-methoxy-5((3,4,5-trimethosyphenyl) seleninyl) phenol (SQ) on human NSCLC (A549 and H460) cells. We found that SQ suppressed the proliferation of NSCLC cells in time- and dose-dependent manners, and blocked the cells at G2/M phase, which was relevant to microtubule depolymerization. Additionally, SQ induced A549 and H460 cell apoptosis by activating the mitochondrial apoptotic pathway. Further, we demonstrated that SQ enhanced the generation of reactive oxygen species (ROS), and pretreatment with N-acetyl- L-cysteine (NAC) attenuated SQ-induced cell apoptosis. Meanwhile, SQ mediated-ROS generation caused DNA damage in A549 and H460 cells. Our data also revealed that SQ-induced apoptosis was correlated with the inhibition of mouse double minute 2 (MDM2) in A549 and H460 cells. In summary, our research indicates that the novel compound SQ has great potential for therapeutic treatment of NSCLC in future.

The anti-MDR efficacy of YAN against A549/Taxol cells is associated with its inhibition on glycolysis and is further enhanced by 2-deoxy-d-glucose.

Aerobic glycolysis is a hallmark of malignant tumor. Here, the hyperactive glycolysis in multidrug-resistant A549/Taxol cells was demonstrated to be essential for maintaining the vigorous cell viability and drug resistance. 5-(4-ethoxyphenyl)-1-(3,4,5-trimethoxyphenyl)-1H-1,2,4-triazol-3-amine (YAN), a newly synthesized tubulin inhibitor, could not only inhibit the glycolysis in A549 and A549/Taxol cells through down-regulating the glycolysis-related proteins, but also disrupt the mitochondrial localization of hexokinase-2 (HK-2) which is related with the apoptosis resistance. The effects of YAN above were relevant to the down-regulation of PI3K-Akt-c-Myc/HIF-1α pathway. Moreover, YAN induced the reactive oxygen species generation in A549 and A549/Taxol cells, which only mediated the apoptosis in A549 cells. We also showed that 2-DG, the glycolysis inhibitor, synergistically enhanced YAN-triggered apoptosis in A549/Taxol cells via further suppressing glycolysis and reducing mitotic slippage. Collectively, we illustrate the inhibition effect of YAN on the glycolysis in A549 and A549/Taxol cells, and provide a fresh insight into the mechanism for the development of YAN as a candidate for multidrug resistant cancer treatment. The finding that 2-DG improved the anti-tumor efficacy of YAN against A549/Taxol cells, offers a reference for solving mitotic slippage-mediated drug resistance.

Novel microtubule inhibitor SQ overcomes multidrug resistance in MCF-7/ADR cells by inhibiting BCRP function and mediating apoptosis.

The occurrence of multidrug resistance (MDR) is one of the impediments in the clinical treatment of breast cancer, and MDR breast cancer has abnormally high breast cancer resistance protein (BCRP/ABCG2) expression. However, there are currently no clinical drugs that inhibit this target. Our previous study found that 2-Methoxy-5((3,4,5-trimethosyphenyl)seleninyl) phenol (SQ0814061/SQ), a small molecule drug with low toxicity to normal tissues, could target microtubules, inhibit the proliferation of breast cancer, and reduce its migration and invasion abilities. However, the effect and the underlying mechanism of SQ on MDR breast cancers are still unknown. Therefore, in this study, we investigated the effect of SQ on adriamycin-resistant MCF-7 (MCF-7/ADR) cells and explored the underlying mechanism. The MTT assay showed that SQ had potent cytotoxicity to MCF-7/ADR cells. In particular, the results of western blot and flow cytometry proved that SQ could effectively inhibit the expression of BCRP in MCF-7/ADR cells to decrease its drug delivery activity. In addition, SQ could block the cell cycle at G2/M phase in parental and MCF-7/ADR cells, thereby mediating cell apoptosis, which was related with the inhibition of PI3K-Akt-MDM2 pathway. Taken together, our findings indicate that SQ overcomes multidrug resistance in MCF-7/ADR cells by inhibiting BCRP function and mediating apoptosis through PI3K-Akt-MDM2 pathway inhibition.

EML4-ALK G1202R mutation induces EMT and confers resistance to ceritinib in NSCLC cells via activation of STAT3/Slug signaling.

The echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase (ALK) fusion gene occurs in approximately 5% of non-small-cell lung cancers (NSCLCs). The development of ALK tyrosine kinase inhibitors (ALK-TKIs) is a major advance in treating NSCLC with the ALK fusion gene. Nevertheless, acquired resistance to ALK-TKIs ultimately limits their use. A prevalent mechanism of drug resistance in kinases occurs through the mutation of G1202R in ALK. However, the mechanisms underlying G1202R resistance to ceritinib are not fully understood. Here, we demonstrated that the expression of EML4-ALK G1202R mutation in A549 cells induced an epithelial-mesenchymal transition (EMT) phenotype and significantly increased the migration and invasion abilities. These phenomena may be due to the upregulation of signal transducer and activator of transcription 3 (STAT3), accompanied by the elevated expression of Slug in EML4-ALK G1202R mutant cells. Furthermore, the combination of ALK and STAT3 inhibitors restored the sensitivity of EML4-ALK G1202R mutant cells to ceritinib. In conclusion, these data indicate that the EML4-ALK G1202R mutation mediates the EMT phenotype by activating the STAT3/Slug signaling pathway, resulting in resistance to ceritinib, and that the combination of STAT3 and ALK inhibitors may overcome ALK mutation-driven drug resistance in the clinic.

Integrated Methylome and Transcriptome Analyses Reveal the Molecular Mechanism by Which DNA Methylation Regulates Kenaf Flowering.

DNA methylation regulates key biological processes in plants. In this study, kenaf seedlings were pretreated with the DNA methylation inhibitor 5-azacytidine (5-azaC) (at concentrations of 0, 100, 200, 400, and 600 µM), and the results showed that pretreatment with 200 µM 5-azaC promoted flowering most effectively. To elucidate the underlying mechanism, phytohormone, adenosine triphosphate (ATP), and starch contents were determined, and genome-wide DNA methylation and transcriptome analyses were performed on anthers pretreated with 200 µM 5-azaC (5-azaC200) or with no 5-azaC (control conditions; 5-azaC0). Biochemical analysis revealed that 5-azaC pretreatment significantly reduced indoleacetic acid (IAA) and gibberellic acid (GA) contents and significantly increased abscisic acid (ABA) and ATP contents. The starch contents significantly increased in response to 200 and 600 µM 5-azaC. Further genome-wide DNA methylation analysis revealed 451 differentially methylated genes (DMGs) with 209 up- and 242 downregulated genes. Transcriptome analysis showed 3,986 differentially expressed genes (DEGs), with 2,171 up- and 1,815 downregulated genes. Integrated genome-wide DNA methylation and transcriptome analyses revealed 72 genes that were both differentially methylated and differentially expressed. These genes, which included ARFs, PP2C, starch synthase, FLC, PIF1, AGL80, and WRKY32, are involved mainly in plant hormone signal transduction, starch and sucrose metabolism, and flowering regulation and may be involved in early flowering. This study serves as a reference and theoretical basis for kenaf production and provides insights into the effects of DNA methylation on plant growth and development.

W436, a novel SMART derivative, exhibits anti-hepatocarcinoma activity by inducing apoptosis and G2/M cell cycle arrest in vitro and in vivo and induces protective autophagy.

Hepatocellular carcinoma (HCC) is considered one of the most common primary liver cancers and the second leading cause of cancer-associated mortality around the world annually. Therefore, it is urgent to develop novel drugs for HCC therapy. We synthesized a novel 4-substituted-methoxybenzoyl-aryl-thiazole (SMART) analog, (5-(4-aminopiperidin-1-yl)-2-phenyl-2H-1,2,3-triazol-4-yl) (3,4,5-trimethoxyphenyl) methanone (W436), with higher solubility, stability, and antitumor activity than SMART against HCC cells in vivo. The purpose of this study was to investigate the mechanisms by which W436 inhibited cell growth in HCC cells. We observed that W436 inhibited the proliferation of HepG2 and Hep3B cells in a dose-dependent manner. Importantly, the anticancer activity of W436 against HCC cells was even higher than that of SMART in vivo. In addition, the antiproliferative effects of W436 on HCC cells were associated with G2/M cell cycle arrest and apoptosis via the activation of reactive oxygen species-mediated mitochondrial apoptotic pathway. W436 also induced protective autophagy by inhibiting the protein kinase B/mammalian target of rapamycin pathway. At the same time, W436 treatment inhibited the cell adhesion and invasion as well as the process of epithelial-to-mesenchymal transition Taken together, our results showed that W436 had the promising potential for the therapeutic treatment of HCC with improved solubility, stability, and bioavailability.

Low dose of fire retardant, 2,2',4,4'-tetrabromodiphenyl ether (BDE47), stimulates the proliferation and differentiation of progenitor Leydig cells of male rats during prepuberty.

2,2',4,4'-Tetrabromodiphenyl ether (BDE47), a flame retardant, is extensively distributed in the food chain. However, whether BDE47 affects Leydig cell development during prepuberty remains unclear. BDE47 was daily gavaged to 21-day-old Sprague-Dawley male rats with 0 (corn oil), 0.1, 0.2, and 0.4 mg/kg for 14 days. BDE47 did not affect the body weight or testis weight of rats. It significantly increased serum testosterone level at 0.4 mg/kg, but decreased luteinizing hormone (LH) level without affecting estradiol level. BDE47 induced Leydig cell hyperplasia (the number of CYP11A1-positive Leydig cells increased), and up-regulated the expression of Scarb1, Star, Hsd11b1, Pcna, and Ccnd1 in the testis. BDE47 significantly reduced p53 and p21 levels but increased CCND1 level. It also markedly increased the phosphorylation of AKT1, AKT2, ERK1/2, and CREB. BDE47 significantly up-regulated the expression of Scarb1, Star, and Hsd11b1 and stimulated androgen production by immature Leydig cells from rats under basal, LH, and 8Br-cAMP stimulated conditions at 100 nM in vitro. In conclusion, BDE47 increased Leydig cell number and up-regulated the expression of Scarb1 and Star, thereby leading to increased testosterone synthesis.

Exposure to 4-bromodiphenyl ether during pregnancy blocks testis development in male rat fetuses.

4-Bromodiphenyl ether (BDE3) is a photodegradation product of higher polybrominated diphenyl ether flame retardants and is known as an endocrine disruptor. However, it is unclear whether and how BDE3 affects the development of fetal testes. This study aimed to investigate the effect of in utero exposure to BDE3 on fetal testicular development in rats. From gestational day (GD) 12-21, BDE3 (0, 50, 100, and 200 mg/kg) was daily gavaged to female pregnant Sprague Dawley rats. BDE3 significantly reduced serum testosterone levels of male pups starting at 50 mg/kg. BDE3 reduced fetal Leydig cell number at a dose of 200 mg/kg without affecting fetal Leydig cell cluster frequency and Sertoli cell number. In addition, BDE3 down-regulated the expression of fetal Leydig cell genes (Cyp11a1, Hsd3b1, Cyp17a1, and Hsd17b3) and their proteins at 100 and/or 200 mg/kg. RNA-seq analysis showed that genes responsive to cAMP (Ass1, Gpd1, Rpl13a) were down-regulated and hypoxia-related genes (Egln3 and P4ha1) were up-regulated at 200 mg/kg. In utero exposure to BDE3 can promote autophagy and apoptosis of fetal Leydig cells via increasing the levels of Beclin1, LC3-II, BAX, and by decreasing the levels of p62 and BCL2. In conclusion, in utero exposure to BDE3 blocks the development of fetal rat testes.

Comparative transcriptomic analysis reveals key genes and pathways in two different cadmium tolerance kenaf (Hibiscus cannabinus L.) cultivars.

Soil cadmium (Cd) contamination has become a massive environmental problem. Kenaf is an industrial fiber crop with high tolerance to heavy metals and could be potentially used for soil phytoremediation. However, the molecular mechanism of Cd in kenaf tolerance remains largely unknown. In the present study, using two contrasting Cd sensitive kenaf (GH and YJ), the key factors accounting for differential Cd tolerance were investigated. GH has a stronger Cd transport and accumulation ability than YJ. In addition, physiological index investigation on malondialdehyde (MDA) contents and antioxidant enzyme (SOD, POD, and CAT) activities showed GH has a stronger detoxification capacity than YJ. Furthermore, the cell ultrastructure of GH is more stable than that of YJ under Cd stress. Transcriptome analysis revealed 2221 (689 up and 1532 down) and 3321 (2451 up and 870 down) genes were differentially expressed in GH and YJ, respectively. More DEGs (differentially expressed genes) were characterized as up-regulated in GH, indicating GH is inclined to activate gene expression to cope with cadmium stress. GO and KEGG analyses indicate that DEGs were assigned and enriched in different pathways. Plenty of critical Cd-induced DEGs such as SOD2, PODs, MT1, DTXs, NRT1, ABCs, CES, AP2/ERF, MYBs, NACs, and WRKYs were identified. The DEGs involved pathways, including antioxidant, heavy metal transport or detoxification, substance transport, plant hormone and calcium signals, ultrastructural component, and a wide range of transcription factors were suggested to play crucial roles in kenaf Cd tolerance, and accounting for the difference in Cd stress sensitivities.

Recent advances of molecular mechanisms of regulating PD-L1 expression in melanoma.

Melanoma is a highly invasive malignant tumor, metastasis can occur in the early stage of the tumor, and the prognosis of patients in the late stage is extremely poor. Programmed cell death protein 1/programmed death-ligand 1 (PD-1/PD-L1) immune checkpoint inhibitors have made a major breakthrough in cancer treatment, which makes the treatment of melanoma enter a new period. The expression of PD-L1 in melanoma is an important biomarker to predict the inhibitory response to the immune checkpoint and is considered to be an independent prognostic indicator of melanoma. Although the related immune checkpoint inhibitors have achieved some good results, the regulation of PD-L1 expression in melanoma is complex and contains multiple types, and few detailed summaries have been done on all types of regulation, so it is very important to explore the complicated regulation mechanism of PD-L1 in melanoma. In this review, we systematically summarize the latest progress on the mechanism of PD-L1 expression regulation in melanoma. The regulatory factors positively related to PD-L1 include internal factors, external induction, signal pathway, transcription factors, epigenetics (Hypomethylation, HDAC6), translation and post-translation levels, while factors negatively related to PD-L1 include microRNA and epigenetics (HDAC8). In addition, the regulation of PD-L1 on the exosome surface is mediated by IFN-γ and there is a positive correlation between them. We hope this review will lay a foundation for the development of more effective and less toxic drugs for immunotherapy of melanoma.

Cisatracurium stimulates testosterone synthesis in rat and mouse Leydig cells via nicotinic acetylcholine receptor.

As a cis-acting non-depolarizing neuromuscular blocker through a nicotinic acetylcholine receptor (nAChR), cisatracurium (CAC) is widely used in anaesthesia and intensive care units. nAChR may be present on Leydig cells to mediate the action of CAC. Here, by Western blotting, immunohistochemistry and immunofluorescence, we identified that CHRNA4 (a subunit of nAChR) exists only on rat adult Leydig cells. We studied the effect of CAC on the synthesis of testosterone in rat adult Leydig cells and mouse MLTC-1 tumour cells. Rat Leydig cells and MLTC-1 cells were treated with CAC (5, 10 and 50 µmol/L) or nAChR agonists (50 µmol/L nicotine or 50 µmol/L lobeline) for 12 hours, respectively. We found that CAC significantly increased testosterone output in rat Leydig cells and mouse MLTC-1 cells at 5 µmol/L and higher concentrations. However, nicotine and lobeline inhibited testosterone synthesis. CAC increased intracellular cAMP levels, and nicotine and lobeline reversed this change in rat Leydig cells. CAC may increase testosterone synthesis in rat Leydig cells and mouse MLTC-1 cells by up-regulating the expression of Lhcgr and Star. Up-regulation of Scarb1 and Hsd3b1 expression by CAC was also observed in rat Leydig cells. In addition to cAMP signal transduction, CAC can induce ERK1/2 phosphorylation in rat Leydig cells. In conclusion, CAC binds to nAChR on Leydig cells, and activates cAMP and ERK1/2 phosphorylation, thereby up-regulating the expression of key genes and proteins in the steroidogenic cascade, resulting in increased testosterone synthesis in Leydig cells.

Neurotrophin-3 stimulates stem Leydig cell proliferation during regeneration in rats.

Neurotrophin-3 (NT-3) acts as an important growth factor to stimulate and control tissue development. The NT-3 receptor, TRKC, is expressed in rat testis. Its function in regulation of stem Leydig cell development and its underlying mechanism remain unknown. Here, we reported the role of NT-3 to regulate stem Leydig cell development in vivo and in vitro. Ethane dimethane sulphonate was used to kill all Leydig cells in adult testis, and NT-3 (10 and 100 ng/testis) was injected intratesticularly from the 14th day after ethane dimethane sulphonate injection for 14 days. NT-3 significantly reduced serum testosterone levels at doses of 10 and 100 ng/testis without affecting serum luteinizing hormone and follicle-stimulating hormone levels. NT-3 increased CYP11A1-positive Leydig cell number at 100 ng/testis and lowered Leydig cell size and cytoplasmic size at doses of 10 and 100 ng/testis. After adjustment by the Leydig cell number, NT-3 significantly down-regulated the expression of Leydig cell genes (Lhcgr, Scarb1, Star, Cyp11a1, Hsd3b1, Cyp17a1, Hsd17b3, Hsd11b1, Insl3, Trkc and Nr5a1) and the proteins. NT-3 increased the phosphorylation of AKT1 and mTOR, decreased the phosphorylation of 4EBP, thereby increasing ATP5O. In vitro study showed that NT-3 dose-dependently stimulated EdU incorporation into stem Leydig cells and inhibited stem Leydig cell differentiation into Leydig cells, thus leading to lower medium testosterone levels and lower expression of Lhcgr, Scarb1, Trkc and Nr5a1 and their protein levels. NT-3 antagonist Celitinib can antagonize NT-3 action in vitro. In conclusion, the present study demonstrates that NT-3 stimulates stem Leydig cell proliferation but blocks the differentiation via TRKC receptor.