Helicobacter pylori Treatment and Gastric Cancer Risk Among Individuals With High Genetic Risk for Gastric Cancer.

Importance: Helicobacter pylori treatment and nutrition supplementation may protect against gastric cancer (GC), but whether the beneficial effects only apply to potential genetic subgroups and whether high genetic risk may be counteracted by these chemoprevention strategies remains unknown. Objective: To examine genetic variants associated with the progression of gastric lesions and GC risk and to assess the benefits of H pylori treatment and nutrition supplementation by levels of genetic risk. Design, Setting, and Participants: This cohort study used follow-up data of the Shandong Intervention Trial (SIT, 1989-2022) and China Kadoorie Biobank (CKB, 2004-2018) in China. Based on the SIT, a longitudinal genome-wide association study was conducted to identify genetic variants for gastric lesion progression. Significant variants were examined for incident GC in a randomly sampled set of CKB participants (set 1). Polygenic risk scores (PRSs) combining independent variants were assessed for GC risk in the remaining CKB participants (set 2) and in an independent case-control study in Linqu. Exposures: H pylori treatment and nutrition supplementation. Main Outcomes and Measures: Primary outcomes were the progression of gastric lesions (in SIT only) and the risk of GC. The associations of H pylori treatment and nutrition supplementation with GC were evaluated among SIT participants with different levels of genetic risk. Results: Our analyses included 2816 participants (mean [SD] age, 46.95 [9.12] years; 1429 [50.75%] women) in SIT and 100 228 participants (mean [SD] age, 53.69 [11.00] years; 57 357 [57.23%] women) in CKB, with 147 GC cases in SIT and 825 GC cases in CKB identified during follow-up. A PRS integrating 12 genomic loci associated with gastric lesion progression and incident GC risk was derived, which was associated with GC risk in CKB (highest vs lowest decile of PRS: hazard ratio [HR], 2.54; 95% CI, 1.80-3.57) and further validated in the analysis of 702 case participants and 692 control participants (mean [SD] age, 54.54 [7.66] years; 527 [37.80%] women; odds ratio, 1.83; 95% CI, 1.11-3.05). H pylori treatment was associated with reduced GC risk only for individuals with high genetic risk (top 25% of PRS: HR, 0.45; 95% CI, 0.25-0.82) but not for those with low genetic risk (HR, 0.81; 95% CI, 0.50-1.34; P for interaction = .03). Such effect modification was not found for vitamin (P for interaction = .93) or garlic (P for interaction = .41) supplementation. Conclusions and Relevance: The findings of this cohort study indicate that a high genetic risk of GC may be counteracted by H pylori treatment, suggesting primary prevention could be tailored to genetic risk for more effective prevention.

Metabolite Alterations and Interactions with Microbiota in Helicobacter pylori-Associated Gastric Lesions.

Metabolites and their interactions with microbiota may be involved in Helicobacter pylori-associated gastric lesion development. This study aimed to explore metabolite alterations upon H. pylori eradication and possible roles of microbiota-metabolite interactions in progression of precancerous lesions. Targeted metabolomics assays and 16S rRNA gene sequencing were conducted to investigate metabolic and microbial alterations of paired gastric biopsy specimens in 58 subjects with successful and 57 subjects with failed anti-H. pylori treatment. Integrative analyses were performed by combining the metabolomics and microbiome profiles from the same intervention participants. A total of 81 metabolites were significantly altered after successful eradication compared to failed treatment, including acylcarnitines, ceramides, triacylglycerol, cholesterol esters, fatty acid, sphingolipids, glycerophospholipids, and glycosylceramides, with P values of <0.05 for all. The differential metabolites showed significant correlations with microbiota in baseline biopsy specimens, such as negative correlations between Helicobacter and glycerophospholipids, glycosylceramide, and triacylglycerol (P < 0.05 for all), which were altered by eradication. The characteristic negative correlations between glycosylceramides and Fusobacterium, Streptococcus, and Gemella in H. pylori-positive baseline biopsy specimens were further noticed in active gastritis and intestinal metaplasia (P < 0.05 for all). A panel including differential metabolites, genera, and their interactions may help to discriminate high-risk subjects who progressed from mild to advanced precancerous lesions in short-term and long-term follow-up periods with areas under the curve (AUC) of 0.914 and 0.801, respectively. Therefore, our findings provide new insights into the metabolites and microbiota interactions in H. pylori-associated gastric lesion progression. IMPORTANCE In this study, a panel was established including differential metabolites, genera, and their interactions, which may help to discriminate high-risk subjects for progression from mild lesions to advanced precancerous lesions in short-term and long-term follow-up.

Allium vegetable intake associated with the risk of incident gastric cancer: a continuous follow-up study of a randomized intervention trial.

BACKGROUND: Allium vegetable components have antibacterial, antioxidative, and immune modulation properties, thus potentially exhibiting antitumor effects. Despite evidence from case-control studies, prospective studies linking allium vegetables with gastric cancer (GC) have been sparse. OBJECTIVE: In a prospective study, we examined whether allium vegetable intake would change the risk of GC occurrence and whether the associations would be modified by vitamin supplementation, garlic supplementation, and Helicobacter pylori (H. pylori) treatment. METHODS: The study was conducted on the basis of the Shandong Intervention Trial, a randomized, placebo-controlled, factorial-designed trial (1995-2003) in a well-recognized high-risk area for GC in China. Participants were continuously followed up to December 2017 for 22.3 y (1995-2017). A total of 3229 subjects were included, with information on the intake of allium vegetables (garlic vegetables and scallions), collected by structured questionnaires in 1994. The associations of total and individual allium vegetable intake with the risk of GC were examined, respectively. RESULTS: During the follow-up, 144 incident cases of GC were identified. Garlic vegetable intake was associated with a decreased risk of incident GC (P-trend = 0.02; OR: 0.83; 95% CI: 0.70, 0.98, per 1 kg/y increment), whereas scallion intake showed no association (P-trend = 0.80). An inverse association of the risk of GC with total allium vegetable and garlic vegetable intake was particularly stronger among those receiving the placebo for vitamin supplementation or garlic supplementation, indicating potential effect modifications by nutritional supplementation on allium vegetable intake and the risk of developing GC. Similar findings were found for analyses of the combined prevalence of dysplasia or GC. CONCLUSIONS: We found a significant reduction in the risk of developing GC with increasing dietary intake of allium vegetables, particularly garlic vegetables. The findings add to the literature on the potential inverse association of garlic vegetable intake with the risk of GC, therefore holding public health implications for dietary recommendations. This trial was registered at clinicaltrials.gov as NCT00339768.

Urine proteomic signatures predicting the progression from premalignancy to malignant gastric cancer.

BACKGROUND: Early detection of gastric cancer (GC) remains challenging. We aimed to examine urine proteomic signatures and identify protein biomarkers that predict the progression of gastric lesions and risk of GC. METHODS: A case-control study was initially designed, covering subjects with GC and gastric lesions of different stages. Subjects were aged 40-69 years, without prior diagnosis of renal or urological diseases. We enrolled a total of 255 subjects, with 123 in the discovery stage from Linqu, China, a high-risk area for GC and 132 in the validation stage from Linqu and Beijing. A prospective study was further designed for a subset of 60 subjects with gastric lesions, which were followed for 297-857 days. FINDINGS: We identified 43 differentially expressed urine proteins in subjects with GC vs. mild or advanced gastric lesions. Baseline urinary levels of ANXA11, CDC42, NAPA and SLC25A4 were further positively associated with risk of gastric lesion progression. Three of them, except for SLC25A4, also had higher expression in GC than non-GC tissues. Integrating these four proteins showed outstanding performance in predicting the progression of gastric lesions (AUC (95% CI): 0.92 (0.83-1.00)) and risk of GC (AUC (95% CI): 0.81 (0.73-0.89) and 0.84 (0.77-0.92) for GC vs. mild or advanced gastric lesions respectively). INTERPRETATION: This study revealed distinct urine proteomic profiles and a panel of proteins that may predict the progression of gastric lesions and risk of GC. These biomarkers in a non-invasive approach may have translational significance for defining high-risk populations of GC and its early detection. FUNDING: Funders are listed in the Acknowledgement.

Plasma lipids signify the progression of precancerous gastric lesions to gastric cancer: a prospective targeted lipidomics study.

Rationale: Gastric cancer (GC) is preceded by a stepwise progression of precancerous gastric lesions. Distinguishing individuals with precancerous gastric lesions that have progression potential to GC is an important need. Perturbated lipid metabolism, particularly the dysregulation of de novo lipogenesis, is involved in gastric carcinogenesis. We conducted the first prospective lipidomics study exploring lipidomic signatures for the risk of gastric lesion progression and early GC. Methods: Our two-stage study of targeted lipidomics enrolled 400 subjects from the National Upper Gastrointestinal Cancer Early Detection Program in China, including 200 subjects of GC and different gastric lesions in the discovery and validation stages. Of validation stage, 152 cases with gastric lesions were prospectively followed for the progression of gastric lesions for a median follow-up of 580 days (interquartile range 390-806 days). We examined the lipidomic signatures associated with the risk of advanced gastric lesions and their progression to GC. Our published tissue proteomic data were referred to further investigate highlighted lipids with their biologically related protein expression in gastric mucosa. Results: We identified 11 plasma lipids significantly inversely associated with the risk of gastric lesion progression and GC occurrence. These lipids were integrated as latent profiles to identify 5 clusters of lipid expression that had distinct risk of gastric lesion progression. The latent profiles significantly improved the ability to predict the progression potential of gastric lesions (AUC: 0.82 vs 0.68, Delong's P = 4.6×10-4) and risk of early GC (AUC: 0.81 vs 0.55, P = 6.3×10-5). Significant associations were found between highlighted lipids, their biologically correlated proteins and the risk of GC, supporting the role of the pathways involving monocarboxylic acid metabolism and lipid transport and catabolic process in GC. Conclusions: Our study revealed the lipidomic signatures associated with the risk of gastric lesion progression and GC occurrence, exhibiting translational implications for GC prevention.

Beneficial effects of endoscopic screening on gastric cancer and optimal screening interval: a population-based study.

BACKGROUND : The effectiveness of endoscopic screening on gastric cancer has not been widely investigated in China and the screening interval of repeated screening has not been determined. METHODS : In a population-based prospective study, we included 375,800 individuals, 14,670 of whom underwent endoscopic screening (2012-2018). We assessed the associations between endoscopic screening and risk of incident gastric cancer and gastric cancer-specific mortality, and examined changes in overall survival and disease-specific survival following screening. The optimal screening interval for repeated endoscopy for early detection of gastric cancer was explored. RESULTS : Ever receiving endoscopic screening significantly decreased the risk of invasive gastric cancer (age- and sex-adjusted relative risk [RR] 0.69, 95 % confidence interval [CI] 0.52-0.92) and gastric cancer-specific mortality (RR 0.33, 95 %CI 0.20-0.56), particularly for noncardia gastric cancer. Repeated screening strengthened the beneficial effect on invasive gastric cancer-specific mortality of one-time screening. Among invasive gastric cancers, screening-detected individuals had significantly better overall survival (RR 0.18, 95 %CI 0.13-0.25) and disease-specific survival (RR 0.18, 95 %CI 0.13-0.25) than unscreened individuals, particularly for those receiving repeated endoscopy. For individuals with intestinal metaplasia or low grade intraepithelial neoplasia, repeated endoscopy at an interval of < 2 years, particularly within 1 year, significantly enhanced the detection of early gastric cancer, compared with repeated screening after 2 years (P-trend = 0.02). CONCLUSION : Endoscopic screening prevented gastric cancer occurrence and death, and improved its prognosis in a population-based study. Repeated endoscopy enhanced the effectiveness. Screening interval should be based on gastric lesion severity.

Proteomic profiling identifies signatures associated with progression of precancerous gastric lesions and risk of early gastric cancer.

BACKGROUND: Molecular features underlining the multistage progression of gastric lesions and development of early gastric cancer (GC) are poorly understood, restricting the ability to GC prevention and management. METHODS: We portrayed proteomic landscape and explored proteomic signatures associated with progression of gastric lesions and risk of early GC. Tissue proteomic profiling was conducted for a total of 324 subjects. A case-control study was performed in the discovery stage (n=169) based on populations from Linqu, a known high-risk area for GC in China. We then conducted two-stage validation, including a cohort study from Linqu (n = 56), with prospective follow-up for progression of gastric lesions (280-473 days), and an independent case-control study from Beijing (n = 99). FINDINGS: There was a clear distinction in proteomic features for precancerous gastric lesions and GC. We derived four molecular subtypes of gastric lesions and identified subtype-S4 with the highest progression risk. We found 104 positively-associated and 113 inversely-associated proteins for early GC, with APOA1BP, PGC, HPX and DDT associated with the risk of gastric lesion progression. Integrating these proteomic signatures, the ability to predict progression of gastric lesions was significantly strengthened (areas-under-the-curve=0.88 (95%CI: 0.78-0.99) vs. 0.56 (0.36-0.76), Delong's P = 0.002). Immunohistochemistry assays and examination at mRNA level validated the findings for four proteins. INTERPRETATION: We defined proteomic signatures for progression of gastric lesions and risk of early GC, which may have translational significance for identifying particularly high-risk population and detecting GC at an early stage, improving potential for targeted GC prevention. FUNDING: The funders are listed in the Acknowledgement.

Dynamic protein expression of NF-κB following rat intracerebral hemorrhage and its association with apoptosis.

The aim of the present study was to evaluate the dynamic protein expression of nuclear factor (NF)-κB and apoptosis in the cerebral tissue surrounding hematoma following intracerebral hemorrhage (ICH) in rats. A total of 80 healthy male Wistar rats were divided into a sham-surgery group and an ICH group. The ICH model was established by injecting autogenous non-heparin anticoagulant arterial blood into the caudate putamen. NF-κB levels were assessed by immunohistochemistry at different time points subsequent to surgery, and apoptosis condition was investigated by terminal deoxynucleotidyl-transferase-mediated dUTP nick end labeling. Different levels of NF-κB were expressed in the cerebral tissue around the ICH at each time point in the ICH group. NF-κB protein expression was detected at 3 h following hemorrhage, mainly in the cytoplasm. Following 6 h, NF-κB was identified in the nucleus. Its expression peaked at 72 h following hemorrhage, and persisted for 5 days. Apoptosis was observed 6 h following hemorrhage, and had increased significantly by 12 h. The rate of apoptosis continued to rise from 72-120 h following hemorrhage. Correlation analysis revealed a significant positive correlation between NF-κB expression and apoptosis (r=0.753; P<0.01). The enhancement of NF-κB expression and apoptosis around ICH, and the significant positive correlation between NF-κB expression and apoptosis, indicates that NF-κB activation may enhance cerebral apoptosis in rats following ICH.

[Mechanism of Duhuo Jisheng decotion in delaying degeneration of nucleus pulposus cells in human intervertebral disc].

This paper aimed to investigate the role of Duhuo Jisheng decotion (DHJSD) in delaying human disc degeneration and its possible molecular mechanism. The intervertebral disc specimens were divided into normal and degenerated groups according to Pfirrmann classfication. The expressions of TNF-α, IL-1ß, MMP-3 and MMP-13 in intervertebral disc tissue were detected by Western blot and PCR. Then degenerated human primary NPCs were cultured in vitro, the viability of NPCs treated with stromal cell-derived factor-1 (SDF-1，10 µg·L⁻¹)and various concentrations of DHJSD was assessed by the CCK-8 assay, and the appropriate concentration was screened. The experiment was divided into three groups, control group, SDF-1 group and DHJSD plus SDF-1 group. The levels of TNF-α, IL-1ß, Agg, coIⅡ, MMP-3 and MMP-13 were detected. The levels of CXCR4, NF-κB major groups P65 phosphorylation (p-P65) and nuclear translocation, after treated with CXCR4 siRNA and NF-κB inhibitor (BAY11-7082) were measured by Western blot and immunofluorescence. At the same time, the expression of cell inflammatory factors and extracellular matrix were also measured. The expressions of TNF-α, IL-1ß, MMP-3 and MMP-13 in the degenerated intervertebral disc tissue were significantly increased. In vitro study, the results of CCK-8 indicated that the viability of NPCs was significantly increased when DHJSD concentration was 300 mg·L⁻¹. After the experiment was divided into three groups, compared with SDF-1 group, the expressions of TNF-α, IL-1ß, MMP-3 and MMP-13 in DHJSD group were significantly decreased, but the expressions of Agg, coIⅡ were significantly increased. When CXCR4-siRNA was transfected into NPCs, SDF-1 increased expressions of CXCR4 and p-P65 and inhibited nuclear translocation of P65, whose effect was suppressed by CXCR4-siRNA and DHJSD. In addition, when BAY11-7082 was used to treat NPCs, the expression of TNF-α, IL-1ß, MMP-3 and MMP-13 were significantly decreased. DHJSD could inhibit the production of inflammatory factors and promote the synthesis of extracellular matrix. The potential mechanism may be related to the SDF-1/CXCR4/NF-κB signaling pathway.

Duhuo Jisheng Decoction inhibits SDF-1-induced inflammation and matrix degradation in human degenerative nucleus pulposus cells in vitro through the CXCR4/NF-κB pathway.

Lower back pain (LBP) is the most common disease in orthopedic clinics world-wide. A classic Fangji of traditional Chinese medicine, Duhuo Jisheng Decoction (DHJSD), has been proven clinically effective for LBP but its therapeutic mechanisms remain unclear. We hypothesized that DHJSD might relieve LBP through inhibiting the exaggerated proinflammatory cytokines and extracellular matrix (ECM) degradation. Thus, we studied the effects of DHJSD on stromal cell-derived factor-1 (SDF-1)-induced inflammation and ECM degradation in human nucleus pulposus cells (hNPCs). The primary hNPCs were isolated from either degenerated human intervertebral disc (HID) of LBP patients or normal HID of lumbar vertebral fracture patients, and cultured in vitro. The cells were treated with SDF-1 (10 ng/mL) and subsequently with different concentrations (100-500 µg/mL) of DHJSD for 24 h, respectively. We found that application of DHJSD significantly antagonized the SDF-1-induced production of proinflammatory cytokines and reduction of aggrecan and type II collagen in the hNPCs. DHJSD also markedly reduced the SDF-1-induced increase of CXCR4 and p-p65 and inhibited the nuclear translocation of p65 in the hNPCs. DHJSD, CXCR4-siRNA, and NF-κB inhibitor (BAY11-7082) caused the same inhibition of exaggerated proinflammatory cytokines in the SDF-1-treated hNPCs. These results provided compelling evidence that DHJSD may inhibit the generation of proinflammatory mediators and ECM degradation of HID through an orchestrated targeting at multiple molecules in the SDF-1/CXCR4/NF-κB pathway, thus offered novel mechanistic insights into the clinical effectiveness of DHJSD on LBP.

Alteration of mitochondrial function and ultrastructure in the hippocampus of pilocarpine-treated rat.

Present studies were carried out to decipher seizure-dependent changes in mitochondrial function and ultrastructure in rat hippocampus after status epilepticus (SE) induced by pilocarpine (PILO). Discernible mitochondrial ultrastructural damage was observed in the hippocampus. Enzyme assay revealed cytochrome oxidase (COX) activity significantly increased 3h after SE, decreased 7 d and 45 d after SE, whereas succinate dehydrogenase (SDH) activity displayed no significant changes. Quantitative real-time PCR and Western blotting showed that COX III (mitochondrial-encoded) mRNA and protein level were significantly increased at 3h, decreased to the control level on 7d and dropped significantly on 45 d; the corresponding expression of COX IV were not changed by PILO at any time tested. The results were also supported by immunohistochemistry. Thus, our results demonstrate that dysfunction of mitochondrial complex IV respiratory enzyme and mitochondrial ultrastructural damage in the hippocampus are associated with prolonged seizure during experimental temporal lobe epilepsy and mitochondria are more vulnerable to epileptic damage.

Houttuyninum, an active constituent of Chinese herbal medicine, inhibits phosphorylation of HER2/neu receptor tyrosine kinase and the tumor growth of HER2/neu-overexpressing cancer cells.

AIMS: The overexpression of HER2/neu receptor plays a key role in tumorigenesis and tumor progression. Small molecules targeting HER2/neu have therapeutic value in cancers that overexpress HER2. In this present study, the effect of houttuyninum, a component in the Chinese herbal medicine Houttuynia cordata Thunb, on HER2/neu tyrosine phosphorylation and its in vivo antitumour activity was investigated. MAIN METHODS: The phosphorylation and expression of proteins were determined by Western blot analysis. The MTT assay was employed to examine the inhibition of cell proliferation in vitro. Xenografts were established in nude mice for evaluating the antitumour activity of houttuyninum in vivo. KEY FINDINGS: Houttuyninum inhibited phosphorylation of HER2 in a dose-dependent manner with an IC50 of 5.52 µg/ml without reducing HER2/neu protein expression in MDA-MB-453 cells. Houttuyninum also inhibited the activation of ERK1/2 and AKT, downstream molecules in the HER2/neu-mediated signal transduction pathway. In contrast, tyrosine phosphorylation of EGFR was unaffected when the concentration of houttuyninum was increased to 40 µg/ml in both A431 cells and MDA-MB-468 cells. Additionally, houttuyninum preferentially inhibited the growth of MDA-MB-453 cells that overexpressed HER2/neu; the MDA-MB-468 cells that overexpress EGFR remained unaffected. Administration of houttuyninum in vivo resulted in a significant reduction of phosphorylated HER2 levels and in tumor volumes of the BT474 and N87 xenografts, which both overexpress HER2/neu. SIGNIFICANCE: Our findings showed that houttuyninum can inhibit the HER2/neu signalling pathway and the tumor growth of cancer cells that overexpress HER2/neu. This drug may provide therapeutic value in the treatment of cancers that involve overexpression of HER2/neu.

SYUNZ-16, a newly synthesized alkannin derivative, induces tumor cells apoptosis and suppresses tumor growth through inhibition of PKB/AKT kinase activity and blockade of AKT/FOXO signal pathway.

Alkannin is the major bioactive compound of Arnebia euchroma roots, which is used in many therapeutic remedies in Chinese traditional medicine. SYUNZ-16 is a new derivative of alkannin. In this study, anticancer effects of SYUNZ-16 on human lung adenocarcinoma cell line GLC-82 and human hepatocarcinoma cell line Hep3B were tested in vitro. The results showed SYUNZ-16 could obviously inhibit the proliferation of these cancer cell lines via induction of apoptosis, with the evidence of increasing AnnexinV-positive cells and cleaved caspase-3 and PARP fragments. More importantly, we found that SYUNZ-16 could inhibit AKT activity in cell-free system. Treatment of cancer cells with SYUNZ-16 decreased the phosphorylation of AKT. Additionally, SYUNZ-16 partially attenuated the phosphorylation levels of FKHR and FKHRL1 in a dose-dependent and time-dependent fashion, and led to an increase in the nuclear accumulation of exogenous FKHR, and upregulated the mRNA expression of Bim and TRADD in cancer cells. Further study showed that constitutively activated AKT1 transfection could reduce apoptosis induction mediated by SYUNZ-16. The in vivo experiments showed that SYUNZ-16 had inhibitory effects on S-180 sarcoma implanted to mice. And in GLC-82 xenograft models, SYUNZ-16 at 20 mg/kg/qod remarkably inhibited the tumor growth with the T/C value of 45.3%. Taken together, SYUNZ-16 might be a potent inhibitor of AKT signaling pathway in tumor cells. These data provide evidence for the development of SYUNZ-16 as a potential antitumor drug candidate for further research and development.

Rhabdastrellic acid-A inhibited PI3K/Akt pathway and induced apoptosis in human leukemia HL-60 cells.

Increasing evidence suggests that aberrant activation of PI3K/Akt is involved in many human cancers, and that inhibition of the PI3K/Akt pathway might be a promising strategy for cancer treatment. Our investigation indicates that Rhabdastrellic acid-A, an isomalabaricane triterpenoid isolated from the sponge, Rhabdastrella globostellata, inhibits proliferation of HL-60 cells with an IC(50) value of 0.68mug/ml, and induces apoptosis. Rhabdastrellic acid-A also induces cleavage of the death substrate poly (ADP-ribose) polymerase (PARP) and caspase-3. Pretreatment of HL-60 cells with the caspase-3 specific inhibitor, DEVD-CHO, prevents Rhabdastrellic acid-A-induced DNA fragmentation and PARP cleavage. Activated PI3K and Akt significantly decreases after treatment with Rhabdastrellic acid-A in HL-60 cells. Expression levels of protein bcl-2, bax remain unchanged in response to Rhabdastrellic acid-A treatment in HL-60 cells. These results suggest that Rhabdastrellic acid-A inhibits PI3K/Akt pathway and induces caspase-3 dependent-apoptosis in HL-60 human leukemia cells.

Synergistic cytotoxicity of Bcl-xL inhibitor, gossypol and chemotherapeutic agents in non-Hodgkin's lymphoma cells.

Anti-apoptotic proteins Bcl-2 and Bcl-xL are overexpressed in 80% of non Hodgkin's lymphoma cells and are thought to play an important role in the resistance of lymphoma cells to current chemotherapeutic agents. Gossypol, an orally-active polyphenolic aldehyde derived from the cotton plant, has been known to have potential anti-neoplastic activity. Recently, gossypol was found to bind to the BH3 binding groove of Bcl-xL and with lesser affinity to Bcl-2. The present study was conducted to determine whether gossypol increases the sensitivity of non-Hodgkin's lymphoma cells to the actions of chemotherapeutic agents by potentiating treatment-induced apoptosis. The interactions observed between gossypol and chemotherapeutic drugs were analyzed using the median effect principle (CalcuSyn analysis). Our data showed that treatment of Ramos cells with gossypol not only induced cell arrest on the G(0)/G(1) phase, but also augmented apoptosis and growth inhibition induced by etoposide (VP-16), doxorubicin hydrochloride (ADM), vincristine (VCR), and paclitaxel (taxol). However, when gossypol was combined with cisplatin (DDP) an antagonistic effect was observed. Gossypol-induced cell cycle arrest was accompanied by decreased expression of cyclin D1 in Ramos cells. In addition, the peroxisome proliferator-activated receptor (PARP) pathway is, at least in part, involved in the gossypol-induced apoptosis when combined with VP-16. These data indicate that single-agent gossypol is effective in inhibiting growth of non-Hodgkin's lymphoma cells in vitro and combination studies with certain secondary chemotherapeutic agents further demonstrate it's synergistic cytotoxicity. These findings support future preclinical and clinical studies of gossypol in the treatment of non-Hodgkin's lymphoma.

[Apoptosis of human leukemia HL-60 cells induced by rhabdastrellic acid-A and its mechanisms].

BACKGROUND & OBJECTIVE: Rhabdastrellic acid-A is an isomalabaricane triterpenoid isolated from the sponge Rhabdastrella globostellata from South China Sea. Our previous study indicated that rhabdastrellic acid-A can inhibit the proliferation of many types of tumor cells with minor toxicity. This study was to investigate the apoptosis of human leukemia HL-60 cells induced by rhabdastrellic acid-A and its possible mechanisms. METHODS: Inhibitory effect of rhabdastrellic acid-A on the proliferation of HL-60 cells was evaluated by MTT assay. DNA fragmentation was analyzed by agarose electrophoresis. Cell morphology was observed under fluorescent microscope. The protein levels of Caspase-3, poly(ADP-ribose) polymerase (PARP), P73, Bcl-2 and Bax were analyzed by Western blot. The expression profile of apoptosis-related genes was analyzed by gene microarray. Reverse transcription-polymerase chain reaction (RT-PCR) was conducted to confirm some altered genes identified by gene microarray. RESULTS: Rhabdastrellic acid-A inhibited the proliferation of HL-60 cells and the 50% inhibition concentration (IC50) was (0.64+/-0.21) microg/ml. When treated with 1 microg/ml rhabdastrellic acid-A for 36 h, condensation of nuclear chromatin of HL-60 cells was observed under fluorescent microscope and DNA fragmentation was observed by agarose electrophoresis. Also, rhabdastrellic acid-A induced cleavage of PARP and Caspase-3. The mRNA levels of 44 genes, including p73, JunD, TNFAIP3 and GADD45A, were up-regulated and the mRNA levels of 16 genes, including MAP2K5 and IGF2R, were down-regulated. The results were further confirmed by RT-PCR. The protein level of P73 was up-regulated after rhabdastrellic acid-A treatment. CONCLUSION: Rhabdastrellic acid-A could induce the apoptosis of HL-60 cells which may be related to the up-regulation of apoptosis-related genes such as p73 and JunD, and the down-regulation of MAP2K5 and IGF2R.

Apoptosis induced by DNA primase inhibitor 3,3'-diethyl-9-methylthia-carbocyanine iodide in human leukemia HL-60 cells.

AIM: To investigate apoptosis induced by 3,3'-diethyl-9-methylthia-carbocyanine iodide (DMTCCI), an inhibitor of DNA primase found in our previous study, and the mechanism of DMTCCI in human myelogenous leukemia HL-60 cells. METHODS: HL-60 cells were cultured in RPMI-1640 medium and treated with different concentrations of DMTCCI. MTT assay was used to detect growth inhibition. Flow cytometry and DNA ladders were used to detect apoptosis. Western blotting was used to observe the expression of survivin, Bcl-xL, Bad, Bax, Bcl-2, caspase-9, caspase-3, caspase-6, PARP, DFF45 and lamin B protein. Caspase-3 activity was measured by ApoAlert Caspase-3 Assay Kit. RESULTS: DMTCCI inhibited proliferation of human leukemia HL-60 cells with IC50 value of 0.24 micromol x L(-1). The results of flow cytometry and DNA ladders showed that DMTCCI could induce apoptosis of HL-60 cells. The expression levels of protein survivin and Bcl-xL were down-regulated, Bad and Bax were up-regulated, while Bcl-2 protein had no change in response to DMTCCI treatment in HL-60 cells. Treatment of HL-60 cells with DMTCCI induced the proteolytic cleavage of caspase-9, caspase-3, caspase-6, PARP, DFF45 and lamin B protein. Caspase-3 activity apparently increased at 3 h and reached a peak at 12 h after exposure to 1 micromol x L(-1) of DMTCCI in HL-60 cells. CONCLUSION: DMTCCI inhibited proliferation and induced apoptosis of human leukemia HL-60 cells. Bcl-2 family proteins, survivin and caspases family proteins might play a role in the apoptosis process induced by DMTCCI.

[Antitumor effect and mechanism of shikonin derivative SYUNZ-7].

BACKGROUND & OBJECTIVE: Natural shikonin compounds and their derivatives have cytotoxicity and antitumor effects. This study was to explore in vitro and in vivo antitumor effects of SYUNZ-7 [2 or 3, 11-bis(phenylsulfanyl)-6-isohexenylnaphthazarin] and the mechanisms. METHODS: In vitro antiproliferation effects of SYUNZ-7 on human lung adenocarcinoma cell line GLC-82, human nasopharyngeal cancer cell line CNE2, human oral cavity cancer cell line KB, human gastric cancer cell line MGC-803 and human hepatocellular cancer cell line HepG2 were tested by MTT assay. In vivo antitumor effect of SYUNZ-7 was tested using ascitic cancer EAC xenograft in mice and CNE2 xenograft in nude mice models. Cell apoptosis and cell cycle distribution were assessed by flow cytometry. The in vivo effect of SYUNZ-7 on angiogenesis was detected by immunohistochemistry. RESULTS: The 50% inhibitory concentrations (IC(50)) of SYUNZ-7 to GLC-82, CNE2, KB, MGC-803, and HepG2 cells were (2.18+/-0.04) microg/ml, (4.17+/-0.09) microg/ml, (5.41+/-0.10) microg/ml, (6.41+/-0.14) microg/ml, and (9.99+/-0.21) microg/ml, respectively. Under the treatment of 1, 2, 4, and 8 mg/kg of SYUNZ-7, the inhibitory rates of EAC xenografts in mice were (40.5+/-0.14)%, (50.9+/-2.3)%, (61.7+/-1.8)%, and (65.6+/-7.4)%, respectively (P<0.01). Under the treatment of 1, 2, and 4 mg/kg of SYUNZ-7, the inhibitory rates of CNE2 xenografts in nude mice were 24.7%, 38.3%, and 41.2%, respectively (P<0.05). SYUNZ-7 induced apoptosis of CNE2 cells in time- and concentration-dependent manners, and blocked the transition of CNE2 cells from S to G(2)/M phase. SYUNZ-7 also inhibited the angiogenesis of CNE2 xenografts in nude mice in a concentration-dependent manner. CONCLUSION: SYUNZ-7 has strong in vivo and in vitro antitumor effects which are related to inducing cell apoptosis, blocking cell cycle, and inhibiting angiogenesis of tumor.

[Inducement effect of Meisoindigo on apoptosis of leukemia cell line HL-60 and its mechanism].

BACKGROUND & OBJECTIVE: Meisoindigo is a powerful drug used in treating chronic myeloid leukemia (CML), but little is known about the mechanisms. This study was to investigate the inducement effect of meisoindigo on apoptosis of myelocytic leukemia cell line HL-60, and explore the possible mechanisms. METHODS: After treatment of meisoindigo, the proliferation of HL-60 cells was detected by trypan blue exclusion assay, and DNA fragmentation by agarose electrophoresis; cell morphology was observed under fluorescent microscope. Cell apoptosis and the expression of Fas were detected by flow cytometry. The expression of Caspase-3, Caspase-8, Caspase-9, PARP, Bcl-2, Bax and the concentration of cytochrome c in cytosol were analyzed by Western blot. RESULTS: Meisoindigo inhibited proliferation and induced apoptosis in HL-60 cells. When treated with 20 micromol/L meisoindigo for 12-48 h, the proliferation of HL-60 cells was significantly inhibited. When treated for 1 h, the apoptosis rate of HL-60 cells was (3.70+/-0.56)%; the apoptosis rate was significantly higher in HL-60 cells treated for 3, 6, and 12 h than in control cells [(19.80+/-1.13)%, (29.20+/-2.69)%, and (47.05+/-7.70)% vs. (2.65+/-0.78)%, P<0.05]. When treated with meisoindigo for 3 h, typical changes of apoptosis, such as chromatin condensation and DNA ladder, were detected in HL-60 cells. The positive rate of Fas was significantly higher in cells treated with 20 micromol/L meisoindigo for 1 h than in control cells [(21.30+/-1.27)% vs. (9.35+/-0.21)%, P<0.05]. Meisoindigo activated Caspase-3, Caspase-8, Caspase-9 and PARP, down-regulated the expression of Bcl-2, up-regulated the expression of Bax and the concentration of cytochrome c. Furthermore, pretreatment of caspase-3 inhibitor z-DEVD-fmk partially reversed the inhibitory effect of meisoindigo on cell proliferation, and decreased cell apoptosis; when treated with meisoindigo for 5 h, the apoptosis rate was significantly higher in pretreated cells than in cells without pretreatment [(29.8+/-5.4)% vs. (16.5+/-5.5)%, P<0.05]; when treated with meisoindigo for 12 h, the alive cell number was significantly lower in pretreated cells than in cells without pretreatment [(1.80+/-0.14) x 10(5)/ml vs. (3.57+/-0.18) x 10(5)/ml, P<0.05]. CONCLUSION: Meisoindigo induces apoptosis of HL-60 cells which may relate to regulation of caspases pathway and bcl-2 family proteins.

[Antitumor and anti-angiogenic effects of manumycin on human hepatocellular carcinoma HepG2 xenografts in nude mice].

BACKGROUND & OBJECTIVE: Farnesyl-transferase inhibitor manumycin has in vitro and in vivo antitumor effects on pancreatic cancer, colon cancer, and anaplastic thyroid carcinoma. Our previous experiments showed that manumycin could inhibit proliferation pathway and survival pathway in human hepatocellular carcinoma HepG2 cells in vitro. This study was to examine the antitumor and anti-angiogenic effects of manumycin on HepG2 xenografts in nude mice. METHODS: The xenografts derived from HepG2 cells were established in BALB/C nude mice. Inoculated mice were randomly divided into normal saline (NS) group, positive control (cyclophosphamide, CTX, 25 mg/kg) group, negative control (0.1% Me2SO, 20 ml/kg) group, low dose munumycin (2.5 mg/kg) group, and high dose munumycin (5 mg/kg) group. Tumor volume was measured in nude mice bearing xenografts. Microvessel density (MVD) was observed by immunohistochemistry. Protein levels of vascular endothelial growth factor (VEGF) and b-fibroblast growth factor (b-FGF) were determined by Western blot; mRNA level of VEGF was analyzed using reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: The mean tumor volume ratio of nude mice xenograft (V/V(0)) was significantly lower in low dose manumycin group and high dose manumycin group than in negative control group (0.68+/-0.09 and 0.59+/-0.04 vs. 1.38+/-0.21, P < 0.01). MVD was significantly lower in manumycin-treated groups than in control group (P < 0.01). Manumycin significantly down-regulated protein level of VEGF in HepG2 cells and HepG2 xenografts, and mRNA level of VEGF in HepG2 xenografts, but didn't affect protein level of b-FGF. CONCLUSIONS: Manumycin could inhibit the growth of human hepatocellular carcinoma HepG2 xenografts in nude mice. The down-regulation of VEGF expression and the inhibition of angiogenesis might play a key role in the anti-neoplastic effect of manumycin.