Gambogic acid exhibits anti-metastatic activity on malignant melanoma mainly through inhibition of PI3K/Akt and ERK signaling pathways.

Gambogic acid (GA) is a potential anti-cancer compound that is extracted from the resin of Garciania hanburyi. The present study was designed to evaluate the anti-metastatic effect of GA on melanoma cell lines in vitro and to explore the underlying mechanism. The anti-proliferative activity of GA on melanoma cells was assessed by CCK-8 assay. The Wound-healing, transwell, adhesion, and tube formation assays were performed to examine the inhibition of GA on the cell's migration, invasion, adhesion, and angiogenesis capacities, respectively. Enzymatic activity of MMP-2 and MMP-9 were detected by gelatin zymography assay. Protein expressions regulated by GA treatment were tested by Western blot assay. The present results showed that GA significantly inhibited the proliferation of highly metastatic melanoma A375, B16-F10 cells and human umbilical vein endothelial cells (HUVECs) in time- and doses-dependent manners. Furthermore, GA significantly inhibited the migratory, invasive and adhesive properties of A375 and B16-F10 cells, and tube-forming potential of HUVECs at sub-IC50 concentrations, where no significant cytotoxicity was observed. Mechanistically, GA treatment suppressed the EMT and angiogenesis processes and reduced the enzymatic activity of MMP-2 and MMP-9. Moreover, abnormal PI3K/Akt and ERK signaling pathways in A375 and B16-F10 cells and HUVECs were notably suppressed by GA treatment. Collectively, our results suggest that GA exerts anti-metastasis activity in melanoma cells by suppressing the EMT and angiogenesis through the PI3K/Akt and ERK signaling pathways, and might be used as a phytomedicine against metastatic melanoma.

Droplet digital PCR for BCR/ABL(P210) detection of chronic myeloid leukemia: A high sensitive method of the minimal residual disease and disease progression.

OBJECTIVE: This study intended to establish a droplet digital PCR (dd-PCR) for monitoring minimal residual disease (MRD) in patients with BCR/ABL (P210)-positive chronic myeloid leukemia (CML), thereby achieving deep-level monitoring of tumor load and determining the efficacy for guided clinically individualized treatment. METHODS: Using dd-PCR and RT-qPCR, two cell suspensions were obtained from K562 cells and normal peripheral blood mononuclear cells by gradient dilution and were measured at the cellular level. At peripheral blood (PB) level, 61 cases with CML-chronic phase (CML-CP) were obtained after tyrosine kinase inhibitor (TKI) treatment and regular follow-ups. By RT-qPCR, BCR/ABL (P210) fusion gene was undetectable in PB after three successive analyses, which were performed once every 3 months. At the same time, dd-PCR was performed simultaneously with the last equal amount of cDNA. Ten CML patients with MR4.5 were followed up by the two methods. RESULTS: At the cellular level, consistency of results of dd-PCR and RT-qPCR reached R2  ≥ 0.99, with conversion equation of Y = 33.148X1.222 (Y: dd-PCR results; X: RT-qPCR results). In the dd-PCR test, 11 of the 61 patients with CML (18.03%) tested positive and showed statistically significant difference (P < .01). In the follow-up of 10 CML patients who were in MR4.5. All patients were loss of MR4.0, and 4 were tested positive by dd-PCR 3 months earlier than by RT-qPCR. CONCLUSION: In contrast with RT-qPCR, dd-PCR is more sensitive, thus enabling accurate conversion of dd-PCR results into internationally standard RT-qPCR results by conversion equation, to achieve a deeper molecular biology-based stratification of BCR/ABL(P210) MRD. It has some reference value to monitor disease progression in clinic.

Oridonin inhibits migration, invasion, adhesion and TGF-ß1-induced epithelial-mesenchymal transition of melanoma cells by inhibiting the activity of PI3K/Akt/GSK-3ß signaling pathway.

Epithelial-mesenchymal transition (EMT) has been reported to play pivotal roles in tumor invasion and metastasis. Inhibition of EMT may exert beneficial effects in regulating metastasis. Oridonin (ORI), an active diterpenoid compound isolated from Rabdosia rubescens, was found to be a potent anti-metastatic agent. However, the possible involvement of ORI in the EMT in malignant melanoma is unclear. The present study found that ORI inhibited cell migration, invasion, and adhesion in A375 and B16-F10 melanoma cells. The transforming growth factor-ß1 (TGF-ß1)-induced EMT was also inhibited in ORI-treated cells, as reflected in the upregulation of E-cadherin, and downregulation of vimentin and Snail. Similar results were observed in A375 and B16-F10 melanoma cells treated with ORI. Furthermore, pre-treatment with ORI blocked the TGF-ß1-induced phosphoinositide 3-kinase (PI3K)/AKT serine/threonine kinase (Akt)/glycogen synthase kinase (GSK)-3ß signaling pathway activation. These effects mimicked PI3 kinase inhibitor LY294002 treatment. ORI interfered with the PI3K/Akt/GSK-3ß pathway, and reversed TGF-ß1-induced EMT, which suppressed the invasion and metastasis of melanoma cells. Taken together, the present study demonstrated that ORI inhibits melanoma cells migration, invasion, and adhesion and TGF-ß1-induced EMT through the PI3K/Akt/GSK-3ß signaling pathway. These findings suggest that ORI is a promising anti-metastasis agent for melanoma.

The prognostic significance of high/positive expression of cyclin B1 in patients with three common digestive cancers: a systematic review and meta-analysis.

Recently, several studies have reported that the expression of cyclin B1 may be associated with the prognosis of cancer. Nevertheless, their conclusions were still controversial. The present was designed to analyze and evaluate the prognostic role of cyclin B1 expression in patients with digestive cancer. PubMed, Embase, Cochrane Library and Web of Science were searched to January, 2017. Pooled odds ratio (OR) with 95% confidence intervals (CIs) were estimated. For the pooled OR estimates of OS, we performed subgroup analysis. Besides, sensitivity analysis was performed to examine the stability of the combined results. All statistical analyses were performed using standard statistical procedures provided in RevMan 5.2. A total of 12 studies (N = 2080 participants) were included for this meta-analysis. The positive/high expression of cyclin B1 had an obvious association with both 3-year overall survival (OR 0.21, 95% CI 0.12-0.37; P < 0.00001) and 5-year overall survival (OR 0.20, 95% CI 0.12-0.34; P < 0.00001) in esophageal cancer, and 5-year overall survival of colorectal cancer (OR 2.01, 95% CI 1.32-3.08; P = 0.001). This meta-analysis indicated that positive/high expression of cyclin B1 may have a close association with worse survival in patients with esophageal cancer, but better prognosis in patients with colorectal cancer.

[mRNA expression of telomere protection protein TIN2 and POT1 in bone marrow of patients with myelodysplastic syndrome].

This study was purposed to explore the relationship between the mRNA expression of telomere protection protein TIN2 and POT1 and the pathogenesis of myelodysplastic syndrome (MDS). The expression of TIN2 and POT1 genes at the mRNA levels were detected by real-time fluorescence quantitative PCR in 51 patients with MDS and 10 normal controls. The results showed that the mRNA expressions of TIN2 in RA/RARS/RCMD/MDS-U, RAEB-1 and RAEB-2 groups according to the World Health Organization criteria were significantly higher than that in the controls (P < 0.05); the mRNA expressions of POT1 in RA/RARS/RCMD/MDS-U, RAEB-1 and RAEB-2 groups were significantly lower than that in the controls (P < 0.05). The mRNA expressions of TIN2 in high-risk group, inter risk-2 group and inter risk-1 group according to the international prognostic scoring system criteria were significantly higher than that in controls (P < 0.05). There was no significant difference between low risk group and the control group. The mRNA expressions of POT1 in high risk group, inter-risk-2 group and inter-risk-1 group were significantly lower than the controls (P < 0.05). There was no significant difference between low risk group and the control group. The mRNA expression of TIN2 in normal chromosome group was significantly lower than that in abnormal chromosome group (P < 0.05). There was no significant difference between normal chromosome group and the control group. The mRNA expression of POT1 in normal chromosome group was significantly higher than that in abnormal chromosome group (P < 0.05). There was no significant difference between normal chromosome group and the control group. It is concluded that the abnormal mRNA expression of TIN2 and POT1 may be involved in the regulation of telomere dynamics of MDS patients, the regulatory mechanism may be related to the telomere length and the pathogenesis of MDS.

[The comparison of three cytogenetic methods for detecting multiple myeloma].

OBJECTIVE: To explore the value of conventional cytogenetic technique (CC), modified cell culture (long term culture and increasing the final concentration of colcemid) and fluorescence in situ hybridization (FISH) in detection of chromosomal and genomic aberrations of multiple myeloma(MM). METHODS: RHG banding was used to evaluate the efficiency of modified culture method on abnormal karyotype detection in 21 MM patients. The probes 1q21, 13q14 (RB1), 14q32 (IGHC/IGHV gene) were used to perform FISH in the detection of chromosomal and genomic aberrations of MM. RESULTS: Abnormal chromosomes were detected in 4 cases (19.1%) of the 21 MM patients with CC. After modified cell culture, abnormal karyotype was detected in 6 cases (28.6%). Abnormal chromosomes were detected in 12 of 18 cases (66.7%) using FISH. Panel FISH disclosed 1q21 amplification in 4 (22.2%), del(13q) abnormality in 5 (27.8%), 14q32 rearrangement in 8 (44.4%). CONCLUSION: FISH can significantly improve the detection rate of chromosomal aberrations in MM.

[Detection of tumor load in chronic myeloid leukemia during treatment with transplantation by conventional cytogenetics, nested-RT-PCR and FISH].

This study was purposed to investigate the sensitivity and specificity of conventional cytogenetics (CC), nested-reverse transcriptase polymerase chain reaction (nested-RT-PCR) and dual-color/dual-fusion fluorescence in situ hybridization (D-FISH) technique in monitoring the tumor load of chronic myeloid leukemia (CML) during treatment with transplantation. CC, nested-RT-PCR and interphase D-FISH were simultaneously carried out to detect the tumor load of 7 CML patients during treatment with non-myeloablative allogeneic stem cell transplantation (allo-NSCT). 40 specimens from 7 CML patients before and after allo-NSCT were analyzed. The results showed that 29 specimens were Ph (+) with different positive ratio and 3 specimens with lower cells were not analyzed by CC. 36 specimens were bcr/abl mRNA (+) by RT-PCR. 4 specimens from case 1 at 12, 18, 26 and 38 months after allo-NSCT were Ph (-) and bcr/abl mRNA (-), 4 Ph (-) bcr/abl (+) specimens containing 2 from case 1 at 9 and 10 months after allo-NSCT, 1 from case 2 at 15 months after allo-NSCT, 1 from case 3 at 12 months after allo-NSCT showed 5.4%, 0%, 16.5% and 1.5% bcr/abl (+) cells by FISH. 3 specimens with lower cells containing 2 from case 5 at 20 and 60 days after allo-NSCT and 1 from case 7 at 40 days after allo-NSCT were analyzed by FISH and showed 55.0%, 27.5% and 73.5% bcr/abl (+) cells. The Ph (-) bcr/abl (-) specimen from case 1 at 12 months post-allo-NSCT showed 0% bcr/abl (+) cells by FISH. It is concluded that CC can be used as a basic tool to monitor the change of tumor load in CML during treatment. When specimen with lower cells can not be analyzed by CC in early period after allo-NSCT, or result of CC can not evaluate precisely dynamic change of tumor load and when tumor load in treated patient are lower to Ph (-) by CC while bcr/abl mRNA (+) by RT-PCR, FISH must be used to detect precisely tumor load and monitor dynamic change of it. More sensitive RT-PCR is used to monitor tumor load when it is lower to bcr/abl (-) by FISH during treatment.

The rate-limiting step in anaerobic digestion in the presence of phosphine.

Methanogenesis is the most important anaerobic biodegradation process in nature, which is accomplished by three different kinds of bacteria - hydrolytic, acetogenic and methanogenic bacteria (MB). An experiment was performed to determine the rate-limiting step of methanogenesis under the influence of various phosphine concentrations (100, 300, 500, 700 and 1000 ppm). It was found that the growth of fermentative bacteria (FB) was severely affected by higher concentrations of phosphine (700 and 971 ppm), while the growth of hydrogen-producing acetogenic bacteria (HPAB) and MB was not affected severely at higher phosphine concentrations. Thus, HPAB and MB are less sensitive to phosphine compared with FB, which means that hydrolysis, and fermentation step is the rate-limiting step during methanogenesis under the influence of phosphine. It is recommended that special attention be paid to the first stage of methanogenesis under high concentrations of phosphine during anaerobic wastewater treatment.

Coexistence of tetrasomy 8 and trisomy 8 in acute promyelocytic leukemia (AML-M3) with t(15;17)(q22;q12).

This study was purposed to characterize the first case of acute promyelocitic leukemia (AML-M(3a)) with t(15;17), trisomy 8 and tetrasomy 8, and explore its characteristics of morphology, cytogenetics, molecular biology, immunology and clinical features. Morphological changes of peripheral blood and bone marrow smears were observed under microscope. Chromosome specimen was prepared by 24 h short-term culture of bone marrow cell, RHG-banding technique was used for karyotypic analysis. PML-RARa fusion gene transcript was detected by nested-reverse transcription-polymerase chain reaction (nested RT-PCR). Interphase fluorescence in situ hybridization (FISH) using chromosome 8 centromere specific probe were carried out to detect abnormal numbers of chromosome 8. Immunophenotypic analysis was performed by flow cytometry. The results showed that peripheral blood smear revealed 65% promyelocyte, and bone marrow aspirate was hypercellular with 72.4% promyelocyte, moderately basophilic cytoplasm with numerous azurophilic granules. Karyotype analysis demonstrated 48, XY, +8, +8, t(15;17)(q22;q12) [16]/47, XY, +8, t(15;17)(q22;q12) [3]/46, XY, t(15;17)(q22;q12) [1]. RT-PCR assay revealed PML-RARa fusion gene transcript (+). FISH showed that the percentages of cells exhibiting 1, 2, 3, 4, 5, 6 green fluorescence signals were 0.5, 7, 19, 55, 18 and 0.5, respectively. This confirmed the presence of tetrasomy 8 and trisomy 8 and also revealed a low percentage of a pentasomy 8 clone. Immunophenotypes of the blasts displayed that CD13 (96.2%), CD33 (55.9%), CYMPO (93.5%) were positive. All the lymphoid markers tested were negative. The patient survival time was just 10 days. It is concluded that tetrasomy 8 is secondary cytogenetic event after t(15;17) in this case. It may be a consequence of clonal evolution of trisomy 8. t(15;17) AML-M(3) with tetrasomy 8 heralds a poor prognosis.

[Detection of abnormal numbers of chromosome 8 with interphase fluorescence in situ hybridization in hematologic malignancies].

OBJECTIVE: To explore the value of interphase fluorescence in situ hybridization (FISH) in the detection of abnormal numbers of chromosome 8 in patients with hematologic malignancies. METHODS: Conventional cytogenetics(CC) and interphase FISH using chromosome 8 centromere specific probe were simultaneously carried out to detect the abnormal numbers of chromosome 8 in eight acute myeloid leukemia cases with CC unveiled abnormal numbers of chromosome 8, ten chronic myeloid leukemia cases in accelerated phase or blast crisis, and three normal individuals. RESULTS: Nine cases that displayed trisomy 8 by means of CC were confirmed by FISH. Among them, Case 5 only displayed diploidy 8, trisomy 8 and tetrasomy 8 by CC, at the same time, FISH confirmed the presence of trisomy 8 and tetrasomy 8 and also revealed a low percentage of a pentasomy 8 clone. Case 3 and Case 17 had each only one cell with trisomy 8 by means of CC, and this could not determine whether they had the trisomy 8 clone, yet FISH confirmed the existence of trisomy 8 clone. Case 9 did not display trisomy 8 by CC, but FISH revealed the existence of trisomy 8 clone. In the other cases, the percentages of trisomy 8 cells determined by FISH were close to or significantly lower than those by CC, but for Case 16 where the percentage of trisomy 8 cells by FISH was significantly higher than that by CC. CONCLUSION: Interphase FISH is a useful method for the detection of abnormal numbers of chromosome 8, especially in the patients with normal or unsure karyotype or with less and bad metaphases. It is an important complement to CC.