Abstract ：

Pyroptosis is an inflammatory form of programmed cell death that plays an important role in regulating tumor progression. Reniformin A (RA) is a natural compound isolated from the medicinal herb Isodon excisoides that has been applied as folk medicine in the treatment of esophageal cancer. However, whether RA has an individual function in cancer and the molecular mechanisms remain unclear. Here, we show that in non-small-cell lung cancer (NSCLC), RA inhibits tumor growth by functioning as a pyroptosis inducer to promote TLR4/NLRP3/ caspase-1/GSDMD axis. Specially, RA treatment increased Toll-like receptor 4 (TLR4) protein expression level by enhancing the TLR4 stability. Based on the molecular docking, we identified that RA directly bound to TLR4 to activate the NLRP3 inflammasome and promote pyroptosis in A549 cells. Moreover, TLR4 is essential for RAinduced pyroptosis, and loss of TLR4 abolished RA-induced pyroptosis and further reduced the inhibitory effect of RA on NSCLC. In vivo experiments confirmed that RA inhibited the growth of lung tumors in mice by affecting pyroptosis in a dose-dependent manner. Furthermore, TLR4 knockdown abolished RA-induced pyroptosis and inhibited the effect of RA chemotherapy in vivo. In conclusion, we propose that RA has a significant anticancer effect in NSCLC by inducing TLR4/NLRP3/caspase-1/GSDMD-mediated pyroptosis, which may provide a potential strategy for the treatment of NSCLC.

Keyword ：

Reniformin A Pyroptosis TLR4 NSCLC

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Background Lithium carbonate (Li2CO3) is widely used in the treatment of clinical-affective psychosis. Exposure to Li2CO3 during pregnancy increases the risk of neural tube defects (NTDs) in offspring, which are severe birth defects of the central nervous system. The mechanism of Li2CO3-induced NTDs remains unclear. Methods C57BL/6 mice were injected with different doses of Li2CO3 intraperitoneally on gestational day 7.5 (GD7.5), and embryos collected at GD11.5 and GD13.5. The mechanisms of Li2CO3 exposure-induced NTDs were determined utilizing immunohistochemistry, western blotting, EdU imaging, enzymatic method, gas chromatography-mass spectrometry (GC-MS), ELISA and HE staining. Results The NTDs incidence was 33.7% following Li2CO3 exposure. Neuroepithelial cell proliferation and phosphohistone H3 level were significantly increased in NTDs embryos, compared with control group (P < 0.01), while the expressing levels of p53 and caspase-3 were significantly decreased. IMPase and GSK-3 beta activity was inhibited in Li2CO3-treated maternal and embryonic neural tissues (P < 0.01 and P < 0.05, respectively), along with decreased levels of inositol and metabolites, compared with control groups (P < 0.01). Conclusions Lithium-induced NTDs model in C57BL/6 mice was established. Enhanced cell proliferation and decreased apoptosis following lithium exposure were closely associated with the impairment of inositol biosynthesis, which may contribute to lithium-induced NTDs. Impact Impairment of inositol biosynthesis has an important role in lithium exposure-induced NTDs in mice model. Lithium-induced NTDs model on C57BL/6 mice was established. Based on this NTDs model, lithium-induced impairment of inositol biosynthesis resulted in the imbalance between cell proliferation and apoptosis, which may contribute to lithium-induced NTDs. Providing evidence to further understand the molecular mechanisms of lithium-induced NTDs and enhancing its primary prevention.

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Tobacco smoke and human papillomavirus (HPV) are both crucial causes of cancer, and their cooperative carcinogenesis has drawn more attention in recent years. Apigenin (AP), a typical flavonoid abundantly found in flowers of plants, vegetables, and fruits, has been demonstrated to exert an anti-carcinogenic effect on various types of cancer. In this study, we investigated the capability of AP against malignant transformation and DNA damage of immortalized human esophageal epithelial (SHEE) cells induced by the synergism of HPV18 and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). The results indicated that the enhancement of migration, invasion, and proliferation ability of SHEE cells induced by HPV and NNK could be effectively inhibited by AP. Moreover, the levels of pyridyloxybutylated (POB)-DNA adducts induced by NNK via P450-catalyzed metabolic activation could also be significantly suppressed by AP. Further analyses on the molecular mechanism revealed that AP inhibited the synergistic carcinogenesis of NNK and HPV on SHEE cells by reducing the expression of mutp53, CDK4, Cyclin D1, and p-Rb (Ser 780), increasing caspase-3 activity, thereby arresting the cell cycle at G1 phase and promoting apoptosis of SHEE cells. We hypothesize that the decrease in NNK-induced POB-DNA adduct levels is related to the deactivation of P450 by AP, which needs to be confirmed in future studies. This study highlights that AP may be employed as a promising chemopreventive agent against cancers in smokers with an HPV infection.

Keyword ：

apigenin human papillomavirus synergistic carcinogenesis chemoprevention 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone tobacco smoke

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Apoptosis is a form of programmed cell death that occurs in multicellular organisms. Fibroblasts are the main cellular ingredients in keloid tissue, which has a relatively low apoptosis level. A natural metabolite of estradiol, 2-Methoxyestradiol (2ME2) exerts a pro-apoptotic effect on tumor cells. In this study, the expression levels of key factors in the apoptosis pathway and the expression level of the proliferating cell nuclear antigen (PCNA) were measured to assess the levels of apoptosis and proliferation in both normal skin fibroblasts and keloid fibroblasts. Twelve samples were obtained from 12 patients: 6 keloid patients and 6 non-keloid patients. All 12 of the patients were randomly selected from the Department of Plastic Surgery at Peking Union Medical College Hospital from June 2016 to December 2016. After cell culture, fibroblasts were divided into the following 6 groups: normal skin fibroblasts (S); keloid fibroblasts (K); keloid fibroblasts treated with 2ME2 (2ME2); keloid fibroblasts treated with DMSO (DMSO); keloid fibroblasts treated with the caspase inhibitor Ac-DEVD-CHO (IN); and keloid fibroblasts treated with both Ac-DEVD-CHO and 2ME2 (IN+2ME2). Fibroblasts at up to passage 3 were used for analysis. Cell activity was measured by the cell counting kit-8. TUNEL staining was used to observe the cell apoptotic morphology. The key apoptosis factors (caspase-3, caspase-8, caspase-9, Bcl-2, Bax, and cytochrome-c) and PCNA expression levels were detected by immunofluorescence analysis and Western blotting. A certain concentration of 2ME2 was also used in group S to evaluate the toxicity. Compared with that in the other groups, 2ME2 significantly inhibited cell activity and led to apoptotic appearance of fibroblasts. In protein analysis, 2ME2 remarkably increased the expression of apoptosis factors and decreased the PCNA expression. Apoptosis levels were reduced by both the caspase inhibitor and 2ME2; thus indicating that the pro-apoptosis effect of 2ME2 was achieved through a caspase-dependent mechanism in keloid fibroblasts. Toxicity assessment showed that 2ME2 had a very low influence on normal skin fibroblasts. 2ME2, considered to be a new promising type of chemotherapy drug, exerts a pro-apoptosis effect by regulating the caspase family and an anti-proliferation effect towards keloid fibroblasts, and it presents low toxicity towards normal fibroblasts in vitro.

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2-Methoxyestradiol apoptosis caspase fibroblast Keloid proliferation

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Purpose: Carboplatin is a first-line treatment for ovarian cancer. However, most patients develop resistance and undergo disease recurrence. This study aims to explore the relationship between the expression of X-linked inhibitor of apoptosis protein ( XIAP) and carboplatin sensitivity in ovarian cancer. Patients and methods: We examined the expression of XIAP in ovarian cancer by immunochemistry. Next, we investigated the role of XIAP in regulating carboplatin sensitivity in ovarian cancer ES2 and 3AO cells through Cell Counting Kit-8 cell viability assay and fluorescein isothiocyanate-Annexin V/propidium iodide apoptosis assay. Expression of apoptotic effectors was measured by Western blot. Results: The immunochemistry results showed that high XIAP expression levels inversely correlated with carboplatin response (P=0.03) and progression-free survival (P=0.0068) in patients with ovarian cancer. Knockdown of XIAP repressed the cell viabilities in the carboplatin-treated cells and increased carboplatin-induced caspase activation. In summary, our data show that XIAP mediates carboplatin sensitivity of ovarian cancer. Conclusion: In summary, our data show that XIAP mediates carboplatin sensitivity of ovarian cancer and XIAP may be a novel target for the treatment of carboplatin-resistant ovarian cancer.

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carboplatin ovarian cancer chemosensitivity XIAP

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We reported two Au clusters with precisely controlled molecular size (Au(5)Peptide(3) and Au(22)Peptide(10)) showing different antitumor effects. In vitro, both Au(5)Peptide(3) and Au(22)Peptide(10) were well taken up by human nasopharyngeal cancer cells (CNE1 cells). However, only Au(5)Peptide(3) significantly induced CNE1 cell apoptosis. Further studies showed that CNE1 cells took up Au(5)Peptide(3) (1.98 X 10(-15) mol/cell), and 9% of them entered mitochondria (0.186 X 10(-15) mol/cell). As a comparison, the uptake of Au(22)peptide(10) was only half the amount of Au(5)Peptide(3) (1.11 X 10(-15) mol/cell), and only 1% of them entered mitochondria (0.016 X 10(-15) mol/cell). That gave 11.6-fold more Au(5)Peptide(3) in mitochondria of CNE1 cells than Au(22)Peptide(10). Further cell studies revealed that the antitumor effect may be due to the enrichment of Au(5)Peptide(3) in mitochondria. Au(5)Peptide(3) slightly decreased the Mcl-1 (antiapoptotic protein of mitochondria) and significantly increased the Puma (pro-apoptotic protein of mitochondria) expression level in CNE1 cells, which resulted in mitochondrial transmembrane potential change and triggered the caspase 9 caspase 3 PARP pathway to induce CNE1 cell apoptosis. In vivo, CNE1 tumor growth was significantly suppressed by Au(5)Peptide(3) in the xenograft model after 3 weeks of intraperitoneal injection. The TUNEL and immuno-histochemical studies of tumor tissue verified that CNE1 cell apoptosis was mainly via the Puma and Mcl-1 apoptosis pathway in the xenograft model, which matched the aforementioned CNE1 cell studies in vitro. The discovery of Au-5 but not Au-22 suppressing tumor growth via the mitochondria target was a breakthrough in the nanomedical field, as this provided a robust approach to turn on/off the nanoparticles' medical properties via atomically controlling their sizes.

Keyword ：

antitumor effect mitochondria peptide-Au cluster Mcl-1 cell apoptosis Puma

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Intracellular invasion and the survival of Staphylococcus aureus in phagocytic cells has been regarded as one of the mechanisms that leads to the treatment failure of S. aureus infection and potential antibiotic resistance. The detection of infected phagocytic cells plays an important role in guiding antibiotic treatment and in reducing drug resistance. The development of a sensitive and specific imaging probe to visualize the intracellular bacteria is quite challenging. In this work, we report a photoacoustic agent (MPC) that is able to detect intracellular S. aureus infection through a dynamic process, including (i) active targeting and internalization into macrophage cells, (ii) specific molecular tailoring by caspase-1 in infected macrophage cells, and (iii) enhancement of the photoacoustic (PA) signal owing to molecular self-assembly. The PA signal per area of the "stimuli-induced assembly" agent (MPC) increases more than 2-fold over that of the active targeting control agent (MPsC). Finally, based on this approach, the average PA signal in the infected site is enhanced by approximately 2.6-fold over that of the control site. We envision that this PA contrast agent may provide a new approach for the selective and sensitive diagnosis of an intracellular bacterial infection.

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bioimaging photoacoustic bacteria Chlorophyll self-assembly

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Apoptosis is a process of programmed cell death that occurs in multicellular organisms. The mitochondrial pathway plays a paramount role in apoptosis. In this study, the expression levels of key factors in the mitochondrial pathway and the cell proliferation factor (PCNA) were measured to evaluate the level of apoptosis and proliferation in keloid scars, physiological scars and normal skin tissue. Thirty samples were taken from 30 patients: 10 keloid patients, 10 physiological scar patients and 10 patients without obvious scarring. All 30 patients were selected randomly from the Department of Plastic Surgery at Peking Union Medical College Hospital from June 2016 to December 2016. Hematoxylin and eosin staining and Masson staining were used to observe the differences in histology and fiber tissue content. Mitochondrial pathway factors (caspase-3, caspase-8, caspase-9, Bcl-2, Bax, cytochrome-c) and PCNA expression levels were detected by immunohistochemistry and were analyzed as the percentage of positively stained cells in the epidermis and dermis. Relative protein expression levels were measured by western blotting. Compared with physiological scars and normal skin tissue, keloid tissue had an increase in fiber number and decrease in cell content. In our immunohistochemical and western blot analyses, all tissue types showed similar expression levels of the mitochondrial pathway factors. However, the percentage of PCNA-positive cells and the relative protein expression level of PCNA were significantly higher in keloid tissue. Keloid has a similar apoptosis level as physiological scars and normal skin but has a higher expression of PCNA, indicating that keloid scars have high levels of proliferation and normal apoptosis.

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proliferation keloid physiological scar skin apoptosis

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Background and objective Blast lung injury is a common type of blast injury and has very high mortality. Therefore, research to identify medical therapies for blast injury is important. Perfluorocarbon (PFC) is used to improve gas exchange in diseased lungs and has antiinflammatory functions in vitro and in vivo. The aim of this study was to determine whether PFC reduces damage to A549 cells caused by blast injury and to elucidate its possible mechanisms of action. Study design and methods A549 alveolar epithelial cells exposed to blast waves were treated with and without PFC. Morphological changes and apoptosis of A549 cells were recorded. PCR and enzymelinked immunosorbent assay (ELISA) were used to measure the mRNA or protein levels of IL-1 beta, IL-6 and TNF-alpha. Malondialdehyde (MDA) levels and superoxide dismutase (SOD) activity levels were detected. Western blot was used to quantify the expression of NF-kappa B, Bax, Bcl-2, cleaved caspase-3 and MAPK cell signaling proteins. Results A549 cells exposed to blast wave shrank, with less cell-cell contact. The morphological change of A549 cells exposed to blast waves were alleviated by PFC. PFC significantly inhibited the apoptosis of A549 cells exposed to blast waves. IL-1 beta, IL-6 and TNF-alpha cytokine and mRNA expression levels were significantly inhibited by PFC. PFC significantly increased MDA levels and decreased SOD activity levels. Further studies indicated that NF-kappa B, Bax, caspase-3, phospho-p38, phosphor-ERK and phosphor-JNK proteins were also suppressed by PFC. The quantity of Bcl-2 protein was increased by PFC.

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Background: In plastic surgery, skin damage induced by ischemia/reperfusion (I/R) is a multifactorial process that often occurs. Methane gas has been reported to be a new therapeutic gas for attenuating I/R injury. In this study, we assessed the effects of methane-rich saline (MRS) in regulating apoptosis on skin flap I/R injury. Methods: Male Sprague-Dawley rats, 6-8 weeks old, were divided randomly into three groups: one sham surgery group (SH) and two surgery groups. After undergoing 6 h of I/R management of an abdominal skin flap, surgery groups were treated with physiological saline (I/R-P) or methane-rich saline (I/R-M). On the 3rd postoperative day, a laser Doppler flowmeter was used to measure flap blood supply, and hematoxylin and eosin (H&E) staining was used to observe morphological changes. TdT-mediated dUTP-X nick end labeling (TUNEL) staining was also used to observe early apoptosis and is presented as the percentage of TUNEL-positive cells. Moreover, pASK-1, pJNK, Bcl-2 and Bax were detected by immunohistochemical technology. Caspase-3 activity was also measured to evaluate the effects of MRS. Results: Compared to the I/R-P group, the flaps in the I/R-M group presented a larger survival area and better blood perfusion with less inflammatory infiltration and cell apoptosis, a higher expression of Bcl-2, a lower expression of pASK-1, pJNK and Bax, and a lower caspase-3 activity. Conclusion: According to the results, MRS attenuated I/R injury by regulating apoptosis and has the potential to be applied as a new therapy for improving skin flap survival.

Keyword ：

Apoptosis Ischemia/reperfusion injury Methane-rich saline Skin flaps

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