p21 promotes gemcitabine tolerance in A549 cells by inhibiting DNA damage and altering the cell cycle.

Gemcitabine is one of the most widely used chemotherapy drugs for advanced malignant tumors, including non-small cell lung cancer. However, the clinical efficacy of gemcitabine is limited due to drug resistance. The aim of the present study was to investigate the role of p21 in gemcitabine-resistant A549 (A549/G+) lung cancer cells. IC50 values were determined using a Cell Counting Kit-8 (CCK-8) assay. mRNA and protein expression levels of genes were measured by reverse transcription-quantitative PCR and western blotting, respectively. The cell cycle distribution and apoptosis rate were analyzed by flow cytometry. DNA damage in cells was evaluated by single-cell gel electrophoresis. The results of western blot analysis and the CCK-8 assay demonstrated that the expression of p21 was higher in A549/G+ cells than in gemcitabine-sensitive cells. Knockdown of p21 expression in gemcitabine-resistant cells sensitized these cells to gemcitabine (with the IC50 decreasing from 84.2 to 26.7 µM). Cell cycle analysis revealed different changes in the cell cycle distribution in A549/G+ cells treated with the same concentration of gemcitabine, and decreased expression of p21 was shown to promote G1 arrest. The apoptosis assay and comet assay results revealed that decreased p21 expression resulted in accumulation of unrepaired DNA double-strand breaks (DSBs) and induction of apoptosis by gemcitabine. The present study demonstrated that knockout of p21 mRNA expression in A549/G+ cells promotes apoptosis and DNA DSB accumulation, accompanied by G1 arrest. These results indicated that p21 is involved in regulating the response of A549 cells to gemcitabine.

MiR-17- 5p/RRM2 regulated gemcitabine resistance in lung cancer A549 cells.

The main objective of this study is to investigate the regulatory roles of the miR-17-5p/RRM2 axis in A549/G+ cells' gemcitabine resistance. The cell viability was determined using CCK8 and clonogenic assays. Gene expression level analysis by RT-qPCR and Western blotting. Cell cycle analysis by flow cytometry. The dual luciferase activity assay was used to verify the target gene of miR-17-5p. In gemcitabine-resistant cell line A549G+, the drug resistance decreased after up-regulation of MiR-17-5p expression. The proportion of cell cycle G1 phase increased, and the S phase decreased. The expression level of cell cycle-related proteins CCNE1, CCNA2, and P21 decreased. The opposite results emerged after the down-regulation of MiR-17-5p expression in gemcitabine-sensitive cell line A549G-. The expression levels of PTEN and PIK3 in A549G+ cells were higher than in A549G-cells, but p-PTEN was lower than that in A549G-. After up-regulating the expression of MiR-17-5p in A549G+, the expression levels of p-PTEN increased, and the expression level of p-AKT decreased. After down-regulating miR-17-5p expression, the opposite results emerged. The dual-luciferase reporter assay and restorative experiments proved that RRM2 is one of the target genes for MiR-17-5p. Our results suggested that the miR-17-5p/RRM2 axis could adjust gemcitabine resistance in A549 cells, and the p-PTEN/PI3K/AKT signal pathway might be involved in this regulatory mechanism.

A novel gut-restricted RIPK1 inhibitor, SZ-15, ameliorates DSS-induced ulcerative colitis.

As a key mediator of cell death and inflammation, receptor-interacting protein kinase 1 (RIPK1) responds to a broad set of inflammatory and pro-death stimuli in human diseases. Inhibitors targeting RIPK1 are being investigated for the treatment of a wide range of human diseases, including ulcerative colitis. In the present study, we designed, synthesized, and investigated the anti-necroptosis and RIPK1-inhibition effects of SZ-15-a symmetrical high-molecular-weight (>500 Da) compound. SZ-15 effectively inhibited necroptosis in U937 and HT-29 cells at concentrations of 1 nM and 10 nM, respectively, and SZ-15 at a concentration of 10 nM almost completely blocked RIPK1, RIPK3, and mixed-lineage kinase domain-like (MLKL) protein phosphorylation induced by necrosis inducers. SZ-15 suppressed the pro-necroptosis function of RIPK1 by downregulating the mRNA expression of pro-inflammatory cytokines, including tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, and IL-6. The activities of SZ-15 were effectively restricted to the gut: The percent recovery of the parent form of SZ-15 in mouse feces was 85.75%. Nevertheless, SZ-15 was effectively absorbed and detected in colon tissues after 1 h at a concentration of 3335 ± 868 ng/g, indicating that membrane permeability was maintained. SZ-15 alleviated dextran sulfate sodium (DSS)-induced ulcerative colitis in vivo by decreasing TNF-α, IL-1ß, IL-22, and IL-6 mRNA expression in colonic tissues. Our preclinical study describes a novel gut-restricted RIPK1 inhibitor that shows great potential for use in the clinical treatment of ulcerative colitis.

An increase in alveolar fluid clearance induced by hyperinsulinemia in obese rats with LPS-induced acute lung injury.

A lower mortality rate is observed in obese patients with acute lung injury (ALI), which is referred to as the obesity paradox, in several studies and recent meta-analyses. Hyperinsulinemia is characterized as the primary effect of obesity, and exogenous insulin attenuates LPS-induced pulmonary edema. The detailed mechanism responsible for the effect of hyperinsulinemia on pulmonary edema and alveolar filling needs to be elucidated. SD rats were fed with a high-fat diet (HFD) for a total of 14 weeks. SD rats were anesthetized and intraperitoneally injected with 10 mg/kg lipopolysaccharide (LPS), while control rats received only saline vehicle. Insulin receptor antagonist S961 (20 nmol/kg) was given by the tail vein and serum, and glucocorticoid-induced protein kinase-1 (SGK-1) inhibitor EMD638683 (20 mg/kg) was administrated intragastrically prior to LPS exposure. The lungs were isolated for the measurement of alveolar fluid clearance. The protein expression of epithelial sodium channel (ENaC) was detected by Western blot. Insulin level in serum was significantly higher in HFD rats compared with normal diet rats in the presence or absence of LPS pretreatment. Hyperinsulinemia induced by high fat feeding increased alveolar fluid clearance and the abundance of α-ENaC, ß-ENaC, and γ-ENaC in both normal rats and ALI rats. Moreover, these effects were reversed in response to S961. EMD638683 prevented the simulation of alveolar fluid clearance and protein expression of ENaC in HFD rats with ALI. These findings suggest that hyperinsulinemia induced by obesity results in the stimulation of alveolar fluid clearance via the upregulation of the abundance of ENaC in clinical acute lung injury, whereas theses effects are prevented by an SGK-1 inhibitor.

Chloroquine increases the anti-cancer activity of epirubicin in A549 lung cancer cells.

The present study investigated whether the autophagy inhibitor chloroquine (CQ) can improve the sensitivity of the A549 lung cancer cell line to epirubicin (EPI). The Cell Counting Kit 8 (CCK8) assay was used to determine the EPI IC50 in A549 cells treated for 72 h. A549 cells were treated with Western blot analysis was performed to detect the expression level of the autophagy-associated protein, microtubule associated protein 1 light chain 3 ß (LC3B), and apoptosis-associated proteins such as cleaved caspase-9 and cleaved caspase-3. CCK8, colony formation, wound healing and Transwell assays were performed to analyze cell proliferation, migration and invasion capacity. Reverse transcription-quantitative PCR (RT-qPCR) was used to analyze the mRNA expression levels of LC3B and beclin-1, and the apoptosis rate was analyzed by flow cytometry. The IC50 of EPI was 0.03 µg/ml. The CCK8 results demonstrated that the cell survival rate was lower in CQ + EPI-treated cells when compared with the individual treatment groups. The colony formation results revealed that the number of clones in the EPI + CQ-treated group was reduced compared with EPI or CQ treatment alone. The wound healing assay revealed that migration was reduced in the EPI + CQ-treated group compared with the other treatment groups, and the Transwell results indicated that the number of cells passing through the Matrigel and membrane was lowest in the CQ + EPI treatment group. The mRNA expression levels of LC3B and beclin-1 were increased in the CQ + EPI group by 51.5 and 61.2%, respectively, when compared with the control group. The results indicated that LC3B protein expression was enhanced by EPI in a concentration-dependent manner, and the protein levels of cleaved caspase-3 and cleaved caspase-9 were higher in the combination group than in the EPI alone group. The flow cytometry results demonstrated that the apoptosis rate was highest in the EPI + CQ group. In conclusion, the autophagy inhibitor CQ increased the sensitivity of A549 cells to EPI, and the underlying mechanism of action may be associated with the activation of apoptosis.

Research progress on the relationship between lung cancer drug-resistance and microRNAs.

Lung cancer, a malignant tumor with the highest death rate of cancer, seriously endangers human health. And its pathogenesis and mechanism of drug resistance has been partially clarified, especially for the signal pathway of epidermal growth factor receptor (EGFR). The targeting therapy of EGFR signaling pathway in non-small cell lung cancer (NSCLC) has achieved a certain effect, but the two mutation of EGFR and other mechanisms of lung cancer resistance still greatly reduce the therapeutic effect of chemotherapy on it. MicroRNA is an endogenous non coding RNA, which has a regulatory function after transcriptional level. Recent studies on the mechanism of lung cancer resistance have found that a variety of microRNAs are related to the mechanism of lung cancer drug-resistance. They can regulate lung cancer resistance by participating in signal pathways, drug resistance genes and cell apoptosis, thus affecting the sensitivity of cancer cells to drugs. Therefore, microRNAs can be used as a specific target for the treatment of lung cancer and plays a vital role in the early diagnosis, prognosis and treatment of lung cancer. This article reviews the mechanisms of lung cancer resistance and its relationship with microRNAs.

Screening miRNAs associated with resistance gemcitabine from exosomes in A549 lung cancer cells.

PURPOSE: To establish a gemcitabine-resistant lung adenocarcinoma cell line, A549/G+, and to screen the differences of miRNA expression in exosomes from A549 and A549/G+ cells. METHODS: A549 cells were exposed in gemcitabine until they were resistant to gemcitabine, and extracted exosomes from A549 and A549/G+. The RNAs from exosomes were subjected to miRNA expression microarray experiments. RESULTS: After 39 weeks of continuous induction, we induced drug resistance in A549 cells. The resistance index was 6. Via GeneChip miRNA 4.0 analysis, there were 446 differential miRNAs between A549 and A549/G+. Target gene prediction and pathway analysis discovered the microRNAs in the intersections may participate in drug resistance. CONCLUSION: These differential miRNAs help to do in-depth research to elucidate the mechanism of resistance to gemcitabine in non-small cell lung cancer.

Effects of baicalin on alveolar fluid clearance and α-ENaC expression in rats with LPS-induced acute lung injury.

Baicalin has been reported to attenuate lung edema in the process of lung injury. However, the effect of baicalin on alveolar fluid clearance (AFC) and epithelial sodium channel (ENaC) expression has not been tested. Sprague-Dawley rats were anesthetized and intratracheally injected with either 1 mg/kg lipopolysaccharide (LPS) or saline vehicle. Baicalin with various concentrations (10, 50, and 100 mg/kg) was injected intraperitoneally 30 min before administration of LPS. Then lungs were isolated for measurement of AFC, cyclic adenosine monophosphate (cAMP) level, and cellular localization of α-ENaC. Moreover, mouse alveolar type II (ATII) epithelial cell line was incubated with baicalin (30 µmol/L), adenylate cyclase inhibitor SQ22536 (10 µmol/L), or cAMP-dependent protein kinase inhibitor (PKA) KT5720 (0.3 µmol/L) 15 min before LPS (1 µg/mL) incubation. Protein expression of α-ENaC was detected by Western blot. Baicalin increased cAMP concentration and AFC in a dose-dependent manner in rats with LPS-induced acute lung injury. The increase of AFC induced by baicalin was associated with an increase in the abundance of α-ENaC protein. SQ22536 and KT5720 prevented the increase of α-ENaC expression caused by baicalin in vitro. These findings suggest that baicalin prevents LPS-induced reduction of AFC by upregulating α-ENaC protein expression, which is activated by stimulating cAMP/PKA signaling pathway.

MiRNA-10b sponge: An anti-breast cancer study in vitro.

Breast cancer is a malignant tumor with the highest incidence among women. Breast cancer metastasis is the major cause of treatment failure and mortality among such patients. MicroRNAs (miRNAs) are a class of small molecular non-coding regulatory RNAs, which act as oncogenes or tumor suppressors in breast cancer. miRNA-10b has been found to exhibit a high expression level in advanced and metastatic breast cancer, and is closely related to breast cancer metastasis. An miRNA sponge is an mRNA with several repeated sequences of complete or incomplete complementarity to the natural miRNA in its 3' non-translating region. It acts as a sponge adsorbing miRNAs and ensures their separation from their targets and inhibits their function. The present study designed a sponge plasmid against miRNA-10b and transiently transfected it into high and low metastatic human breast cancer cell lines MDA-MB-231 and MCF-7, and analyzed the effects of the miRNA-10b sponge on the growth and proliferation, migration and invasion in these cell lines. qRT-PCR results found that the sponge plasmid effectively inhibited the expression of miRNA-10b, and upregulated the expression of the miRNA­10b target protein HOXD-10. The results from the CCK-8 assay found that the miRNA-10b sponge inhibited the growth of breast cancer cell lines MDA-MB-231 and MCF-7. Results of the plate cloning experiments indicated that the miRNA-10b sponge suppressed the colony formation of the MDA-MB-231 and MCF-7 cells. The results of wound healing and Transwell assays showed that the miRNA-10b sponge inhibited the migration and invasion of the breast cancer cell lines MDA-MB-231 and MCF-7. Our results demonstrated that the miRNA-10b sponge effectively inhibited the growth and proliferation of breast cancer MDA-MB-231 and MCF-7 cells. In addition, it also restrained the migration and invasion of human highly metastatic breast cancer MDA-MB-231 cells.

Bifunctional fused polypeptide inhibits the growth and metastasis of breast cancer.

Breast cancer is the most common cancer and the leading cause of cancer-related death among women worldwide, with urgent need to develop new therapeutics. Targeted therapy is a promising strategy for breast cancer therapy. Stromal-derived factor-1/CXC chemokine receptor 4 (CXCR4) has been implicated in the metastasis of breast cancer, which renders it to be therapeutic target. This study aimed to evaluate the anticancer effect of fused TAT- DV1-BH3 polypeptide, an antagonist of CXCR4, and investigate the underlying mechanism for the cancer cell-killing effect in the treatment of breast cancer in vitro and in vivo. This results in a potent inhibitory effect of fused TAT-DV1-BH3 polypeptide on tumor growth and metastasis in nude mice bearing established MDA-MB-231 tumors. Fused TAT-DV1-BH3 polypeptide inhibited the proliferation of MDA-MB-231 and MCF-7 cells but did not affect that of HEK-293 cells. The fused TAT-DV1-BH3 polypeptide colocalized with mitochondria and exhibited a proapoptotic effect through the regulation of caspase-9 and -3. Furthermore, the fused TAT-DV1-BH3 polypeptide suppressed the migration and invasion of the highly metastatic breast cancer cell line MDA-MB-231 in a concentration-dependent manner. Notably, the DV1-mediated inhibition of the stromal-derived factor-1/CXCR4 pathway contributed to the antimetastasis effect, evident from the reduction in the level of phosphoinositide 3 kinase and matrix metalloproteinase 9 in MDA-MB-231 cells. Collectively, these results indicate that the apoptosis-inducing effect and migration- and invasion-suppressing effect explain the tumor regression and metastasis inhibition in vivo, with the involvement of caspase- and CXCR4-mediated signaling pathway. The data suggest that the fused TAT-DV1-BH3 polypeptide is a promising agent for the treatment of breast cancer, and more studies are warranted to fully elucidate the therapeutic targets and molecular mechanism.

Fused polypeptide with DEF induces apoptosis of lung adenocarcinoma cells.

To analyze the effects of a new unknown peptide DEF on the growth of tumor cells, a fused polypeptide TAT-DV1-DEF was designed and synthesized. The lung adenocarcinoma cell line GLC-82 treated with TAT- DV1-DEF was analyzed with a cell counting kit 8, and the location of polypeptides in cells was observed under laser confocal microscopy. The efficiency of polypeptide transfection and changes in nuclear morphology were analyzed by flow cytometry and fluorescence microscopy, respectively. Finally, the mechanism of tumor cell growth inhibition was evaluated by Western blotting. We found that TAT-DV1-DEF could significantly inhibit the growth of the lung adenocarcinoma cell line GLC-82, but not the normal human embryonic kidney cell line HEK-293. Polypeptides were found to be mostly localized in the cytoplasm and some mitochondria. The efficiency of polypeptide transfection in the two cell types was approximately 99%. Apoptotic nuclei were observed under fluorescence microscopy upon treatment with polypeptides and DAPI staining. Western blot analyses indicated that the polypeptide inhibition of tumor cell growth was apoptosis dependent. In the present study, we demonstrated that fused polypeptides could induce apoptosis of the lung adenocarcinoma cell line GLC-82, indicating that the new unknown peptide DEF has antitumor effects.

Protective effects of fucoxanthin against ferric nitrilotriacetate-induced oxidative stress in murine hepatic BNL CL.2 cells.

Fucoxanthin is a carotenoid that is rich in some seaweed. Although fucoxanthin has been reported to possess radical-scavenging activities in vitro, little is known whether it may protect against iron-induced oxidative stress in cultured cells. In this study, we examined the protection of fucoxanthin against oxidative damage in BNL CL.2 cells induced by ferric nitrilotriacetate (Fe-NTA). The data show that incubation of BNL CL.2 cells with Fe-NTA for 30 min significantly decreased cell proliferation, whereas pretreatment with fucoxanthin (1-20 µΜ) for 24h significantly recovered cell proliferation in a dose-dependent manner. In addition, fucoxanthin pretreatment significantly decreased intracellular reactive oxygen species (ROS) and DNA damage in BNL CL.2 cells incubated with Fe-NTA for 30 min. Moreover, fucoxanthin markedly decreased the level of thiobarbituric acid-reactive substances (TBARS) and protein carbonyl contents in BNL CL.2 cells induced by Fe-NTA. By contrast, fucoxanthin significantly increased the levels of GSH in a concentration-dependent manner. These results demonstrate that fucoxanthin at 1-20µΜ effectively prevents cytotoxicity in BNL CL.2 cells treated with Fe-NTA, and that the protective effect is likely associated with decreased intracellular ROS, TBARS, protein carbonyl contents and increased GSH levels.