Celastrol acts synergistically with afatinib to suppress non-small cell lung cancer cell proliferation by inducing paraptosis.

Non-small cell lung cancer (NSCLC) with wild-type epidermal growth factor receptor (EGFR) is intrinsic resistance to EGFR-tyrosine kinase inhibitors (TKIs), such as afatinib. Celastrol, a natural compound with antitumor activity, was reported to induce paraptosis in cancer cells. In this study, intrinsic EGFR-TKI-resistant NSCLC cell lines H23 (EGFR wild-type and KRAS mutation) and H292 (EGFR wild-type and overexpression) were used to test whether celastrol could overcome primary afatinib resistance through paraptosis induction. The synergistic effect of celastrol and afatinib on survival inhibition of the NSCLC cells was evaluated by CCK-8 assay and isobologram analysis. The paraptosis and its modulation were assessed by light and electron microscopy, Western blot analysis, and immunofluorescence. Xenografts models were established to investigate the inhibitory effect of celastrol plus afatinib on the growth of the NSCLC tumors in vivo. Results showed that celastrol acted synergistically with afatinib to suppress the survival of H23 and H292 cells by inducing paraptosis characterized by extensive cytoplasmic vacuolation. This process was independent of apoptosis and not associated with autophagy induction. Afatinib plus celastrol-induced cytoplasmic vacuolation was preceded by endoplasmic reticulum stress and unfolded protein response. Accumulation of intracellular reactive oxygen species and mitochondrial Ca2+ overload may be initiating factors of celastrol/afatinib-induced paraptosis and subsequent cell death. Furthermore, Celastrol and afatinib synergistically suppressed the growth of H23 cell xenograft tumors in vivo. The data indicate that a combination of afatinib and celastrol may be a promising therapeutic strategy to surmount intrinsic afatinib resistance in NSCLC cells.

[MRI/TRUS cognitive fusion combined with 12-core systematic transperineal prostate biopsy for the diagnosis of clinically significant prostate cancer: A report of 208 cases].

OBJECTIVE: To investigate the detection rate and complications of magnetic resonance imaging / transrectal ultrasonography (MRI/TRUS) cognitive fusion combined with 12-core systematic transperineal prostate biopsy (TPPB) in the diagnosis of clinically significant PCa (CS-PCa). METHODS: This retrospective study included 208 patients undergoing first-time MRI/TRUS cognitive fusion combined with 12-core systematic TPPB from June 2015 to May 2019. The patients, aged 54－85 (67.6 ± 7.8) years, all received digital rectal examination, PSA detection, TRUS and prostate multiparametric MRI (mpMRI) before biopsy. We analyzed the mpMRI images, identified and marked the suspected signal areas, repeated TRUS for further observation of the prostate, conducted cognitive fusion based on the mpMRI images and determined the target before 12-core systematic TPPB and subjecting the samples obtained to pathological examination. RESULTS: Of the 208 patients, 112 were diagnosed with CS-PCa (no case with tPSA < 4 µg/L, 21 cases with 4 µg/L ≤ tPSA < 10 µg/L, 47 cases with 10 µg/L ≤ tPSA < 20 µg/L, 40 cases with 20 µg/L ≤ tPSA < 100 µg/L, and 4 cases with tPSA ≥ 100 µg/L), 85 with BPH, 8 with chronic prostatitis, 2 with atypical prostatic hyperplasia, and 1 with prostatic intraepithelial neoplasia. Systemic inflammatory response syndrome occurred in 3 and gross hematuria and/or bloody stool in 12 cases after biopsy, which were all cured by anti-infection and hemostasis treatment. CONCLUSIONS: MRI/TRUS cognitive fusion combined with 12-core systematic transperineal prostate biopsy can improve the detection rate of the initial diagnosis of clinically significant PCa with a low incidence of controllable complications.

PARP inhibitor resistance: the underlying mechanisms and clinical implications.

Due to the DNA repair defect, BRCA1/2 deficient tumor cells are more sensitive to PARP inhibitors (PARPi) through the mechanism of synthetic lethality. At present, several PAPRi targeting poly (ADP-ribose) polymerase (PARP) have been approved for ovarian cancer and breast cancer indications. However, PARPi resistance is ubiquitous in clinic. More than 40% BRCA1/2-deficient patients fail to respond to PARPi. In addition, lots of patients acquire PARPi resistance with prolonged oral administration of PARPi. Homologous recombination repair deficient (HRD), as an essential prerequisite of synthetic lethality, plays a vital role in killing tumor cells. Therefore, Homologous recombination repair restoration (HRR) becomes the predominant reason of PARPi resistance. Recently, it was reported that DNA replication fork protection also contributed to PARPi resistance in BRCA1/2-deficient cells and patients. Moreover, various factors, such as reversion mutations, epigenetic modification, restoration of ADP-ribosylation (PARylation) and pharmacological alteration lead to PARPi resistance as well. In this review, we reviewed the underlying mechanisms of PARP inhibitor resistance in detail and summarized the potential strategies to overcome PARPi resistance and increase PARPi sensitivity.

Curcumin reverses cisplatin resistance in cisplatin-resistant lung caner cells by inhibiting FA/BRCA pathway.

Cisplatin (DDP) is the most widely used chemotherapy agent for treatment of malignancies including lung cancer. However, the effectiveness of DDP is often weakened by acquired resistance of tumor cells. DDP kills cancer cells primarily by creating intrastrand and interstrand DNA cross-links, which block DNA replication. The Fanconi anemia (FA)/BRCA pathway is a DNA cross-link damage repair pathway, which regulates cellular resistance to DNA cross-link agents, such as DDP. Some study has shown that natural compound curcumin sensitize human ovarian and breast cancer cells to DDP. However, whether curcumin may reverse resistance to DDP in DDP-resistant lung cancer cells has not been understood. In this study, we showed that curcumin enhanced the proliferation inhibitory effect of DDP and promote DDP-induced apoptosis in A549/DDP cells (DDP-resistant lung adenocarcinoma cells). Moreover, we observed that FA/BRCA pathway DNA damage repair processes, such as DDP-induced FANCD2 monoubiquitination and nuclear foci formation were downregulated in the presence of curcumin, suggesting that curcumin enhanced sensitivity to DDP in A549/DDP cells through the inhibition of FA/BRCA pathway. Furthermore, the calculation of q value and apoptosis analyses revealed that curcumin in combination with DDP could exert a synergistic cytotoxic effect in A549/DDP cells, further demonstrating that curcumin can reverse cisplatin resistance of A549/DDP cells. In conclusion, by suppressing the FA/BRCA pathway DNA repair, curcumin potentiates DDP-induced proliferation inhibitory effect and apoptosis in A549/DDP cell, indicating that curcumin may serve as a chemosensitizer to cross-link-inducing anticancer drugs DDP.

RNA interferences targeting the Fanconi anemia/BRCA pathway upstream genes reverse cisplatin resistance in drug-resistant lung cancer cells.

BACKGROUND: Cisplatin is one of the most commonly used chemotherapy agent for lung cancer. The therapeutic efficacy of cisplatin is limited by the development of resistance. In this study, we test the effect of RNA interference (RNAi) targeting Fanconi anemia (FA)/BRCA pathway upstream genes on the sensitivity of cisplatin-sensitive (A549 and SK-MES-1) and -resistant (A549/DDP) lung cancer cells to cisplatin. RESULT: Using small interfering RNA (siRNA), knockdown of FANCF, FANCL, or FANCD2 inhibited function of the FA/BRCA pathway in A549, A549/DDP and SK-MES-1 cells, and potentiated sensitivity of the three cells to cisplatin. The extent of proliferation inhibition induced by cisplatin after knockdown of FANCF and/or FANCL in A549/DDP cells was significantly greater than in A549 and SK-MES-1 cells, suggesting that depletion of FANCF and/or FANCL can reverse resistance of cisplatin-resistant lung cancer cells to cisplatin. Furthermore, knockdown of FANCL resulted in higher cisplatin sensitivity and dramatically elevated apoptosis rates compared with knockdown of FANCF in A549/DDP cells, indicating that FANCL play an important role in the repair of cisplatin-induced DNA damage. CONCLUSION: Knockdown of FANCF, FANCL, or FANCD2 by RNAi could synergize the effect of cisplatin on suppressing cell proliferation in cisplatin-resistant lung cancer cells through inhibition of FA/BRCA pathway.

Prognostic value of EpCAM/MUC1 mRNA-positive cells in non-small cell lung cancer patients.

The aim of this study was to assess the prognostic value of EpCAM/MUC1 mRNA-positive circulating tumor cells (CTCs) in patients with non-small cell lung cancer (NSCLC). The presence of EpCAM/MUC1 mRNA-positive CTCs was evaluated in 74 NSCLC patients before the initiation of any therapy, from which 61 patients with surgical resection of tumor were also evaluable for EpCAM/MUC1 mRNA-positive CTC analysis after surgery, by quantitative real-time PCR assay. Sixty patients with benign lung disease (BLD) entered this study as controls. The results showed that blood levels of EpCAM and MUC1 mRNA in NSCLC patients before and after surgery were significantly higher than those in BLD patients (P = 0.001 and P = 0.015, respectively, for EpCAM; P = 0.003 and P = 0.026, respectively, for MUC1), and the levels of the two gene mRNA in NSCLC patients significantly decreased after surgery (P = 0.025 and P = 0.033, respectively). Disease recurrence significantly increased in NSCLC patients with EpCAM/MUC1 mRNA-positive CTC preoperation and postoperation (P = 0.004 and P = 0.001, respectively). Disease-free survival and overall survival significantly reduced in patients with EpCAM/MUC1 mRNA-positive CTC preoperation and postoperation (P = 0.012 and P = 0.002, respectively, for preoperation; both P < 0.001 for postoperation). Multivariate analysis demonstrated that the presence of EpCAM/MUC1 mRNA-positive CTCs before and after surgery was an independent factor associated with disease recurrence. In conclusion, the detection of EpCAM/MUC1 mRNA-positive CTCs in the blood before and after surgery is useful for predicting a poor prognosis in NSCLC patients who undergo curative surgery.

Loss of SHROOM3 affects neuroepithelial cell shape through regulating cytoskeleton proteins in cynomolgus monkey organoids.

Neural tube defects (NTDs) are severe congenital neurodevelopmental disorders arising from incomplete neural tube closure. Although folate supplementation has been shown to mitigate the incidence of NTDs, some cases, often attributable to genetic factors, remain unpreventable. The SHROOM3 gene has been implicated in NTD cases that are unresponsive to folate supplementation; at present, however, the underlying mechanism remains unclear. Neural tube morphogenesis is a complex process involving the folding of the planar epithelium of the neural plate. To determine the role of SHROOM3 in early developmental morphogenesis, we established a neuroepithelial organoid culture system derived from cynomolgus monkeys to closely mimic the in vivo neural plate phase. Loss of SHROOM3 resulted in shorter neuroepithelial cells and smaller nuclei. These morphological changes were attributed to the insufficient recruitment of cytoskeletal proteins, namely fibrous actin (F-actin), myosin II, and phospho-myosin light chain (PMLC), to the apical side of the neuroepithelial cells. Notably, these defects were not rescued by folate supplementation. RNA sequencing revealed that differentially expressed genes were enriched in biological processes associated with cellular and organ morphogenesis. In summary, we established an authentic in vitro system to study NTDs and identified a novel mechanism for NTDs that are unresponsive to folate supplementation.

Identification and validation of an immune-related lncRNAs signature to predict the overall survival of ovarian cancer.

Ovarian cancer (OC) is the most lethal gynecological cancer in women. Studies had reported that immune-related lncRNAs signatures were valuable in predicting the survival and prognosis of patients with various cancers. In our study, the prognostic value of immune-related lncRNAs was investigated in OC patients from TCGA-RNA-seq cohort (n=378) and HG-U133_Plus_2 cohort (n=590), respectively. Pearson correlation analysis was implemented to screen the immune-related lncRNA and then univariate Cox regression analysis was performed to explore their prognostic value in OC patients. Five prognostic immune-related lncRNAs were identified as prognostic lncRNAs. Besides, they were inputted into a LASSO Cox regression to establish and validate an immune-related lncRNA prognostic signature in TCGA-RNA-Seq cohort and HG-U133_Plus_2 cohort, respectively. Based on the best cut-off value of risk score, patients were divided into high- and low-risk groups. Survival analysis suggested that patients in the high-risk group had a worse overall survival (OS) than those in the low-risk group in both cohorts. The association between clinicopathological feathers and risk score was then evaluated by using stratification analysis. Moreover, we constructed a nomogram based on risk score, age and stage, which had a strong ability to forecast the OS of the OC patients. The influence of risk score on immune infiltration and immunotherapy response were assessed and the results suggested that patients with high-risk score might recruit multiple immune cells and stromal cells, leading to facilitating immune surveillance evasive. Ultimately, we demonstrated that the risk model was associated with chemotherapy response of multiple antitumor drugs, especially for paclitaxel, metformin and veliparib, which are commonly used in treating OC patients. In conclusion, we constructed a novel immune-related lncRNA signature, which had a potential prognostic value for OC patients and might facilitate personalized counselling for immunotherapy and chemotherapy.

Efficacy of the FOLFOX/CAPOX regimen for advanced small bowel adenocarcinoma: a three-center study from China.

PURPOSE: Small bowel adenocarcinoma (SBA) is a rare malignancy, and most patients present with unresectable or metastatic disease. Thus far, no standard chemotherapeutic regimen has been established and related data are scarce, especially in Eastern countries. The purpose of this multicenter study was to evaluate the efficacy and toxicity of oxaliplatin combined with fluoropyrimidines in Chinese patients with advanced SBA. METHODS: Advanced SBA patients who received FOLFOX (5-fluorouracil/5-FU plus leucovorin and oxaliplatin)/ CAPOX (capecitabine plus oxaliplatin) as first-line chemotherapy between February, 2004 and October, 2010 were identified from 3 large medical centers in China. The response rate (RR), progression-free survival (PFS), overall survival (OS), and chemotherapy-associated toxicity were evaluated. Cox models were applied for multivariate analyses. RESULTS: Of 34 patients, with SBA 28 received FOLFOX and 6 CAPOX. The objective response (OR) and disease control (DC) rates were 32.3% and 61.7%, respectively. The median PFS and OS were 6.3 and 14.2 months, respectively. The toxicity was tolerable, grade 3-4 toxicity was rare. In multivariate analysis, only multi-organ metastasis reached borderline significance for shorter PFS (p=0.059), but the variables of age (>65 years; p=0.001), and multi-organ metastasis (p=0.001) were significantly associated with poor OS. CONCLUSION: To our knowledge, this multicenter retrospective study is the first and largest one among Asian studies at present estimating oxaliplatin combined with fluoropyrimidines as first-line chemotherapy for advanced SBA. FOLFOX/CAPOX is proved effective for advanced SBA in this study, but the results do not absolutely agree with previous studies from Western countries, showing that further studies are still needed.

Development of a novel transcription factors-related prognostic signature for serous ovarian cancer.

Growing evidence suggest that transcription factors (TFs) play vital roles in serous ovarian cancer (SOC). In the present study, TFs mRNA expression profiles of 564 SOC subjects in the TCGA database, and 70 SOC subjects in the GEO database were screened. A 17-TFs related prognostic signature was constructed using lasso cox regression and validated in the TCGA and GEO cohorts. Consensus clustering analysis was applied to establish a cluster model. The 17-TFs related prognostic signature, risk score and cluster models were effective at accurately distinguishing the overall survival of SOC. Analysis of genomic alterations were used to elaborate on the association between the 17-TFs related prognostic signature and genomic aberrations. The GSEA assay results suggested that there was a significant difference in the inflammatory and immune response pathways between the high-risk and low-risk score groups. The potential immune infiltration, immunotherapy, and chemotherapy responses were analyzed due to the significant difference in the regulation of lymphocyte migration and T cell-mediated cytotoxicity between the two groups. The results indicated that patients with low-risk score were more likely to respond anti-PD-1, etoposide, paclitaxel, and veliparib but not to gemcitabine, doxorubicin, docetaxel, and cisplatin. Also, the prognostic nomogram model revealed that the risk score was a good prognostic indicator for SOC patients. In conclusion, we explored the prognostic values of TFs in SOC and developed a 17-TFs related prognostic signature to predict the survival of SOC patients.

Effect of nitric oxide on the proliferation of AGS gastric cancer cells.

BACKGROUND AND OBJECTIVE: Nitric oxide (NO) is involved in many physiologic and pathologic processes. As an important biologic mediator, NO has been the focus of cancer study for its function in tumorigenesis, tumor progression, and death. This study investigated the effect of NO donor sodium nitroprusside (SNP) on the growth and proliferation of gastric cancer cell line AGS. METHODS: The growth inhibition of AGS cells was analyzed using MTT assay. The cell cycle was measured using flow cytometry. The changes of mRNA expression of proliferating cell nuclear antigen (PCNA) and caspase-3 were examined using reverse transcriptase polymerase chain reaction (RT-PCR), and the protein expressions of PCNA and caspase-3 were analyzed using Western blot. RESULTS: Dose-dependent SNP inhibited cell growth and proliferation. When the AGS cells were treated with SNP at 100, 500, 1000, 1500, and 2000 mumol/L for 24 h, the growth inhibition rates were (2.02 +/- 2.96)%, (10.82 +/- 2.21)%, (18.95 +/- 3.35)%, (26.88 +/- 2.54)%, and (42.57 +/- 1.27)%, respectively (P < 0.05). SNP altered the cell cycle in AGS cells. Compared with the control group, treatment with SNP at 100, 500, 1000, 1500, and 2000 mumol/L for 24 h reduced the number of cells in the S phase by 2.29%, 7.8%, 11.34%, 20.49%, and 23.6%, respectively, and enhanced the number of cells in the G1/G0 phases by 3.33%, 9.3%, 13.46%, 21.37%, and 24.73%, respectively (P < 0.05). With the increasing concentration and action time of SNP, the expressions of PCNA mRNA and protein decreased. The expression of caspase-3 mRNA remained unchanged, but procaspase-3 was activated. CONCLUSION: NO not only inhibits cell growth and proliferation, but also induces apoptosis in gastric cancer cells, and such effects of NO showed significant dose-dependent activity.

Mechanisms of metformin inhibiting cancer invasion and migration.

Cancer currently ranks among the leading causes of death globally. Cancer invasion and metastasis transform locally grown cancers to a systemic and life-threatening disease, which accounts for the most significant challenge in cancer treatment. Recent studies showed that Metformin, the most commonly used first-line oral drug for the treatment of type 2 diabetes (T2DM), could prevent and treat various cancers. Moreover, multiple evidence suggested that metformin inhibited cancer invasion and metastasis, which could improve the prognosis of cancer patients administrated with metformin. To better understand the anti-cancer role of metformin, the present review summarized the potential mechanisms of inhibiting cancer invasion and metastasis by metformin, including AMPK signaling pathway, EMT signaling pathway, epigenetic modification and so on. However, multiple problems remain unresolved and more clinical trials are needed to prove the inhibition of cancer invasion and metastasis by metformin.

Increased attention to snake images in cynomolgus monkeys: an eye-tracking study.

Previous studies have revealed faster detection of snake images in humans and non-human primates (NHPs), suggesting automatic detection of evolutionary fear-relevant stimuli. Furthermore, human studies have indicated that general fear-relevance rather than evolutionary relevance is more effective at capturing attention. However, the issue remains unclarified in NHPs. Thus, in the present study, we explored the attentional features of laboratory-reared monkeys to evolutionary and general fear-relevant stimuli (e.g., images of snakes, capturing gloves). Eye-tracking technology was utilized to assess attentional features as it can provide more accurate latency and variables of viewing duration and frequency compared with visual search task (VST) and response latency adopted in previous studies. In addition, those with autism spectrum disorder (ASD) show abnormal attention to threatening stimuli, including snake images. Rett syndrome (RTT) is considered a subcategory of ASD due to the display of autistic features. However, the attentional features of RTT patients or animal models to such stimuli remain unclear. Therefore, we also investigated the issue in MECP2 gene-edited RTT monkeys. The influence of different cognitive loads on attention was further explored by presenting one, two, or four images to increase stimulus complexity. The eye-tracking results revealed no significant differences between RTT and control monkeys, who all presented increased viewing (duration and frequency) of snake images but not of aversive stimuli compared with control images, thus suggesting attentional preference for evolutionary rather than general fear-relevant visual stimuli. Moreover, the preference was only revealed in visual tasks composed of two or four images, suggesting its cognitive-load dependency.

Potential use of actigraphy to measure sleep in monkeys: comparison with behavioral analysis from videography.

Sleep is indispensable for human health, with sleep disorders initiating a cascade of negative consequences. As our closest phylogenetic relatives, non-human primates (NHPs) are invaluable for comparative sleep studies and exhibit tremendous potential for improving our understanding of human sleep and related disorders. Previous work on measuring sleep in NHPs has mostly used electroencephalography or videography. In this study, simultaneous videography and actigraphy were applied to observe sleep patterns in 10 cynomolgus monkeys ( Macaca fascicularis) over seven nights (12 h per night). The durations of wake, transitional sleep, and relaxed sleep were scored by analysis of animal behaviors from videography and actigraphy data, using the same behavioral criteria for each state, with findings then compared. Here, results indicated that actigraphy constituted a reliable approach for scoring the state of sleep in monkeys and showed a significant correlation with that scored by videography. Epoch-by-epoch analysis further indicated that actigraphy was more suitable for scoring the state of relaxed sleep, correctly identifying 97.57% of relaxed sleep in comparison with video analysis. Only 34 epochs (0.13%) and 611 epochs (2.30%) were differently interpreted as wake and transitional sleep compared with videography analysis. The present study validated the behavioral criteria and actigraphy methodology for scoring sleep, which can be considered as a useful and a complementary technique to electroencephalography and/or videography analysis for sleep studies in NHPs.

Localization and translocation of RhoA protein in the human gastric cancer cell line SGC-7901.

AIM: To elucidate the localization of RhoA in gastric SGC-7901 cancer cells and its translocation by lysophosphatidic acid (LPA) and/or 8-chlorophenylthio-cAMP (CPT-cAMP). METHODS: Immunofluorescence microscopy was used to determine the localization of RhoA. Western blotting was used to detect both endogenous and exogenous RhoA in different cellular compartments (membrane, cytosol, nucleus) and the translocation of RhoA following treatment with LPA, CPT-cAMP, or CPT-cAMP + LPA. RESULTS: Immunofluorescence staining revealed endogenous RhoA to be localized in the membrane, the cytosol, and the nucleus, and its precise localization within the nucleus to be the nucleolus. Western blotting identified both endogenous and exogenous RhoA within different cellular compartments (membrane, cytosol, nucleus, nucleolus). After stimulation with LPA, the amount of RhoA within membrane and nuclear extracts increased, while it decreased in the cytosol fractions. After treatment with CPT-cAMP the amount of RhoA within the membrane and the nuclear extracts decreased, while it increased within the cytosol fraction. Treatment with a combination of both substances led to a decrease in RhoA in the membrane and the nucleus but to an increase in the cytosol. CONCLUSION: In SGC-7901 cells RhoA was found to be localized within the membrane, the cytosol, and the nucleus. Within the nucleus its precise localization could be demonstrated to be the nucleolus. Stimulation with LPA caused a translocation of RhoA from the cytosol towards the membrane and the nucleus; treatment with CPT-cAMP caused the opposite effect. Furthermore, pre-treatment with CPT-cAMP was found to block the effect of LPA.

Breviscapine, a traditional Chinese medicine, alleviates myocardial ischaemia reperfusion injury in diabetic rats.

OBJECTIVE: The aim of this study was to explore the effects of breviscapine on the expressions of intercellular adhesion molecule-I (ICAM-I), ATPase activities and oxidative stress in ischaemia-reperfused (I/R) myocardium of diabetic rats. METHODS: Sprague Dawley rats were randomly divided into two groups (a diabetic group and a non-diabetic group), and each group divided into two subgroups including a control group and a breviscapine group. Reperfusion surgery was carried out in all rats.The contents of malonaldehyde (MDA), superoxide dismutase (SOD) and glutathione peroxidase (GSH-P(x)) in serum and myocardial tissues were measured. The activities of Na(+)-K(+)-ATPase, Mg(2+)-ATPase, Ca(2+)-ATPase in myocardial mitochondria were measured. The ICAM-I protein expressions in myocardium were determined with the immunohistochemical method. RESULTS: The activities of Na(+)-K(+)-ATPase, Mg(2+)-ATPase, Ca(2+)-ATPase were significantly increased in diabetic rats in the breviscapine group compared with the control group. Compared with the non-diabetic control group, the contents of MDA in serum and myocardium were significantly increased in the diabetic control group. Breviscapine led to a reduction of the contents of MDA in the diabetic and non-diabetic group. Compared with the non-diabetic control group, the activities of SOD and GSH-P(x) in the myocardium were significantly decreased in the diabetic control group.The activities of SOD and GSH-P(x) in serum and myocardium were increased in the diabetic and non-diabetic group after breviscapine treatment. Compared with the non-diabetic control group, the ICAM- I protein expressions were increased significantly in the diabetic control group. Breviscapine decreased the ICAM-I protein expression in the diabetic and the non-diabetic group. CONCLUSION: Breviscapine may have protective effects on myocardial ischaemia reperfusion injury of diabetic rats by scavenging oxygen free radicals, decreasing the expressions of ICAM-I protein in myocardium and increasing the activities of Na(+)-K(+)-ATPase, Mg(2+)-ATPase, Ca(2+)-ATPase in myocardial mitochondria.

[Association of RAS mutations in circulating cell-free DNA in the plasma with clinicopathological features of colorectal cancer].

OBJECTIVE: To detect RAS mutations in the circulating cell-free DNA (cfDNA) in the plasma and explore the their correlation with the clinicopathological features in patients with colorectal cancer. METHODS: Real-time PCR was used to detect RAS mutations in plasma cfDNA and matched tumor tissue DNA samples from 71 colorectal cancer patients. The correlation of RAS mutations with the clinicopathological features of the patients were analyzed. RESULTS: Of the 71 patients with colorectal cancer, 23 (32.39%) showed RAS mutations in the cfDNA and 36 (50.7%) showed RAS mutations in tumor tissue DNA, with a concordance rate of 76.06% in the results between the two samples (Kappa=0.523). RAS mutations in the cfDNA were not related to the patients' age (P=0.072), gender (P=0.320), tumor stage (IVa and IVb, P=0.450), primary tumor position (P=0.324), lung metastasis (P=0.237), CEA level (P=0.284) or CA199 level (P=0.427). The positivity rate of RAS mutations in plasma cfDNA was significantly higher in patients with liver metastasis than those without liver metastasis (P=0.045). CONCLUSION: Plasma cfDNA can be a reliable source of diagnostic DNA to replace the tumor tissue DNA for diagnosis of RAS mutations. RAS mutations in plasma cfDNA occur more frequently in colorectal cancer patients with liver metastasis.

Phosphorylated vasodilator-stimulated phosphoprotein is localized on mitotic spindles of the gastric cancer cell line SGC-7901.

AIM: To elucidate the localization of vasodilator stimulated phosphoprotein (VASP), a cytoskeletal organizing protein and a substrate of protein kinases A and G in mitotic gastric cancer cells. METHODS: Immunofluorescence microscopy was used to observe the localization of alpha-tubulin, VASP and Ser157 phosphorylated VASP (p-VASP) in interphase of mitotic gastric cancer of the cell line SGC-7901. RESULTS: Immunofluorescence staining showed that p-VASP but not VASP was co-localized with alpha-tubulin on spindle poles and fibers in prophase, metaphase and anaphase of the mitotic process of the gastric cancer cell line SGC-7901. H89, an inhibitor of protein kinases A and G, had no effect on the localization of p-VASP on the spindles. CONCLUSION: VASP may play a role in assembling and stabilizing the mitotic spindle of cells, and phosphorylation of the protein is the precondition for it to exert this function.

H pylori stimulates proliferation of gastric cancer cells through activating mitogen-activated protein kinase cascade.

AIM: To explore the mechanism by which H pylori causes activation of gastric epithelial cells. METHODS: A VacA (+) and CagA (+) standard H pylori line NCTC 11637 and a human gastric adenocarcinoma derived gastric epithelial cell line BGC-823 were applied in the study. MTT assay and (3)H-TdR incorporation test were used to detect the proliferation of BGC-823 cells and Western blotting was used to detect the activity and existence of related proteins. RESULTS: Incubation with H pylori extract increased the proliferation of gastric epithelial cells, reflected by both live cell number and DNA synthesis rate. The activity of extracellular signal-regulated protein kinase (ERK) signal transduction cascade increased within 20 min after incubation with H pylori extract and appeared to be a sustained event. MAPK/ERK kinase (MEK) inhibitor PD98059 abolished the action of H pylori extract on both ERK activity and cell proliferation. Incubation with H pylori extract increased c-Fos expression and SRE-dependent gene expression. H pylori extract caused phosphorylation of several proteins including a protein with molecular size of 97.4 kDa and tyrosine kinase inhibitor genistein inhibited the activation of ERK and the proliferation of cells caused by H pylori extract. CONCLUSION: Biologically active elements in H pylori extract cause proliferation of gastric epithelial cells through activating tyrosine kinase and ERK signal transduction cascade.

Co-inhibition of pol θ and HR genes efficiently synergize with cisplatin to suppress cisplatin-resistant lung cancer cells survival.

Cisplatin exert its anticancer effect by creating intrastrand and interstrand DNA cross-links which block DNA replication and is a major drug used to treat lung cancer. However, the main obstacle of the efficacy of treatment is drug resistance. Here, we show that expression of translesion synthesis (TLS) polymerase Q (POLQ) was significantly elevated by exposure of lung cancer cells A549/DR (a cisplatin-resistant A549 cell line) to cisplatin. POLQ expression correlated inversely with homologous recombination (HR) activity. Co-depletion of BRCA2 and POLQ by siRNA markedly increased sensitivity of A549/DR cells to cisplatin, which was accompanied with impairment of double strand breaks (DSBs) repair reflected by prominent cell cycle checkpoint response, increased chromosomal aberrations and persistent colocalization of p-ATM and 53BP1 foci induced by cisplatin. Thus, co-knockdown of POLQ and HR can efficiently synergize with cisplatin to inhibit A549/DR cell survival by inhibiting DNA DSBs repair. Similar results were observed in A549/DR cells co-depleted of BRCA2 and POLQ following BMN673 (a PARP inhibitor) treatment. Importantly, the sensitization effects to cisplatin and BMN673 in A549/DR cells by co-depleting BRCA2 and POLQ was stronger than those by co-depleting BRCA2 and other TLS factors including POLH, REV3, or REV1. Our results indicate that there is a synthetic lethal relationship between pol θ-mediated DNA repair and HR pathways. Pol θ may be considered as a novel target for lung cancer therapy.