Kang-Ai Injection Inhibits Gastric Cancer Cells Proliferation through IL-6/STAT3 Pathway / 中国结合医学杂志

OBJECTIVE@#To explore the mechanisms underlying the proliferative inhibition of Chinese herbal medicine Kang-Ai injection (KAI) in gastric cancer cells.@*METHODS@#Gastric cancer cell lines MGC803 and BGC823 were treated by 0, 0.3%, 1%, 3% and 10% KAI for 24, 48 and 72 h, respectively. The cell proliferation was evaluated by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay. The apoptosis and cell cycle were evaluated by flow cytometry. Interleukin (IL)-6 mRNA and protein expression levels were detected by quantitative real-time polymerase chain reaction (qRT-PCR) and enzyme-linked immune sorbent assay (ELISA), respectively. The protein expression levels of cyclin A, cyclin E, cyclin B1, cyclin D1, p21, retinoblastoma (RB), protein kinase B (AKT), extracellular regulated protein kinases (ERK), signal transducer and activator of transcription (STAT) 1 and STAT3 were detected by Western blot.@*RESULTS@#KAI inhibited the proliferation of MGC803 and BGC823 gastric cancer cells in dose- and time-dependent manner. After treated with KAI for 48 h, the proportion of G1 phase was increased, expression level of cyclin D1 and phosphorylation-RB were down-regulated, whereas the expression of p21 was up-regulated (all P<0.01). Furthermore, 48-h treatment with KAI decreased the phosphorylation level of STAT3, inhibited the mRNA and protein expressions of IL-6 (all P<0.01). IL-6 at dose of 10 ng/mL significantly attenuated the proliferative effect of both 3% and 10% KAI, and recovered KAI-inhibited STAT3 phosphorylation and cyclin D1 expression level (all P<0.01).@*CONCLUSION@#KAI exerted an anti-proliferative function by inhibiting IL-6/STAT3 signaling pathway followed by the induction of G1 phase arrest in gastric cancer cells.

Protective autophagy antagonizes oxaliplatin-induced apoptosis in gastric cancer cells / 癌症

Oxaliplatin-based chemotherapy is used for treating gastric cancer. Autophagy has been extensively implicated in cancer cells; however, its function is not fully understood. Our study aimed to determine if oxaliplatin induce autophagy in gastric cancer MGC803 cells and to assess the effect of autophagy on apoptosis induced by oxaliplatin. MGC803 cells were cultured with oxaliplatin. Cell proliferation was measured using MTT assay, and apoptosis was determined by flow cytometry. Protein expression was detected by Western blot. Autophagy was observed using fluorescent microscopy. Our results showed that the rate of apoptosis was 9.73% and 16.36% when MGC803 cells were treated with 5 and 20 μg/mL oxaliplatin for 24 h, respectively. In addition, caspase activation and poly ADP-ribose polymerase (PARP) cleavage were detected. Furthermore, when MGC803 cells were treated with oxaliplatin for 24 h, an accumulation of punctate LC3 and an increase of LC3-II protein were also detected, indicating the activation of autophagy. Phosphorylation of Akt and mTOR were inhibited by oxaliplatin. Compared to oxaliplatin alone, the combination of autophagy inhibitor chlorochine and oxaliplatin significantly enhanced the inhibition of cell proliferation and the induction of cell apoptosis. In conclusion, oxaliplatin-induced protective autophagy partially prevents apoptosis in gastric cancer MGC803 cells. The combination of autophagy inhibitor and oxaliplatin may be a new therapeutic option for gastric cancer.

Cisplatin enhances TRAIL-induced apoptosis in gastric cancer cells through clustering death receptor 4 into lipid rafts / 中华肿瘤杂志

<p><b>OBJECTIVE</b>Gastric cancer cells are insensitive to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). To sensitize gastric cancer cells to TRAIL, we treated gastric cancer MGC803 cells with TRAIL and cisplatin.</p><p><b>METHODS</b>Cell proliferation was measured using MTT assay. Cell apoptosis was determined by flow cytometry. Expression of proteins was analyzed by Western blot. The distribution of lipid rafts and death receptors was analyzed by immunofluorescence microscopy. MGC803 cells were pretreated with 50 mg/L nystatin for 1 h, and followed by the treatment of cisplatin and TRAIL.</p><p><b>RESULTS</b>100 µg/L TRAIL resulted in (8.51 ± 3.45)% inhibition of cell proliferation and caused (3.26 ± 0.89)% cell apoptosis in MGC803 cells. Compared with the treatment with cisplatin alone, treatment with TRAIL (100 µg/L) and cisplatin (8.49 mg/L, IC(50) dose of 24 h) led to a dramatic increase in both inhibition of cell proliferation [(52.58 ± 4.57)% vs. (76.43 ± 5.35)%, P < 0.05] and cell apoptosis [(23.10 ± 3.41)% vs. (42.56 ± 4.11)%, P < 0.05]. Moreover, cleavage of caspase-8 and caspase-3 was detected. TRAIL (100 µg/L) did not induce obvious lipid rafts aggregation and death receptor 4 (DR4) clustering, while cisplatin (8.49 mg/L) significantly promoted the localization of DR4 in aggregated lipid rafts. Pretreatment with 50 mg/L nystatin, a cholesterol-sequestering agent, triggered (3.66 ± 0.52)% cell apoptosis after 24 h. Pretreatment with nystatin for 1 h before the addition of 8.49 mg/L cisplatin for 24 h caused a decreased tendency to cell apoptosis [(25.74 ± 3.28)% vs. (22.76 ± 2.97)%]. While, pretreatment with nystatin before the addition of cisplatin and TRAIL, the proportion of apoptotic cells decreased from (43.16 ± 4.26)% to (31.52 ± 3.99)% (P < 0.05).</p><p><b>CONCLUSION</b>Cisplatin enhances TRAIL-induced apoptosis in gastric cancer MGC803 cells through clustering death receptors into lipid rafts.</p>

Expression of c-Cbl, Cbl-b, and epidermal growth factor receptor in gastric carcinoma and their clinical significance / 癌症

<p><b>BACKGROUND AND OBJECTIVE</b>c-Cbl and Cbl-b are two ubiquitous members of the Casitas B-lineage lymphoma (Cbl) family, which play important roles in the downregulation of epidermal growth factor receptor (EGFR) by acting as E3 ubiquitin ligases and multiadaptor proteins. This study investigated the expression of c-Cbl, Cbl-b, and EGFR in gastric carcinoma and its clinical significance.</p><p><b>METHODS</b>The expressions of c-Cbl, Cbl-b, and EGFR were detected by immunohistochemistry using tissue microarrays consisting of 124 specimens of gastric carcinoma and 16 specimens of normal gastric mucosa. The relationship between the expressions of c-Cbl, Cbl-b, and EGFR and clinicopathologic factors of gastric carcinoma were analyzed statistically.</p><p><b>RESULTS</b>The positive rates of c-Cbl, Cbl-b, and EGFR were higher in the gastric carcinoma group than in the normal group (71.0% vs. 18.0%, P<0.01; 82.3% vs. 25.0%, P<0.01; 56.5% vs. 12.5%, P<0.01, respectively). The expression of c-Cbl was positively correlated with depth of invasion (r=0.219, P=0.015), and TNM staging (r=0.266, P=0.003). The expression of Cbl-b was positively correlated with lymph node metastasis (r=0.190, P<0.034) and TNM staging (r=0.298, P<0.001). The expression of EGFR was positively correlated with depth of invasion (r=0.286, P<0.001) and TNM staging (r=0.362, P=0.000). The expression of both c-Cbl and Cbl-b was positively correlated with EGFR (r=0.241, P=0.007; r=0.183, P=0.042, respectively). Synchronous strong-positive expressions of c-Cbl, Cbl-b, and EGFR were observed in 27 specimens of gastric carcinoma, most of which were at advanced stage.</p><p><b>CONCLUSIONS</b>Overexpressions of c-Cbl, Cbl-b, and EGFR are closely related to the invasion and progression of gastric carcinoma. c-Cbl and Cbl-b may serve as novel molecular markers for gastric carcinoma.</p>

Effects of Bufalin on SYK and CBL family proteins in induction of HL-60 cell apoptosis / 中国实验血液学杂志

The study was aimed to explore the mechanism of SYK and CBL family of ubiquitin ligases in Bufalin-induced HL-60 cells apoptosis. Cell viability was tested by trypan blue staining and apoptosis was detected by using flow cytometry. The expressions of CBL and CBL-b and the phosphorylation of SYK were detected by using immunoprecipitation and Western blot. The results showed that Bufalin inhibited HL-60 cell proliferation in time- and dose-dependent manners. IC(50) of suppressing cell viability at 24, 48 and 72 hours were about 26.3, 7.8 and 2.0 nmol/L respectively. The high dose of bufalin already induced apoptosis of HL-60 cells at 8 hours. SYK was quickly phosphorylated, and the expressions of CBL and CBL-b were down-regulated after treatment with Bufalin. It is concluded that SYK activation and CBL protein down-regulation may be involved in Bufalin-induced HL-60 cell apoptosis.

Effect of mitogen-activated protein kinases on ATRA-induced differentiation of NB4 cells / 中国实验血液学杂志

The aim of this study was to observe the effect of p38MAPK inhibitor SB203580 on ATRA-induced differentiation of NB4 cells. The proliferation activity of cells was assayed by MTT method, the cell cycle was detected by flow cytometry, the differentiation of NB4 cells into granulocytes was measured by test of NBT reduction, the activity of extracellular signal-regulated kinase (ERK) was detected by substrate phosphorylation. The results showed that the ATRA in 0.01-01 micromol/L inhibited the proliferation of NB4 cells in time-and dose-dependent manner and induced the differentiation of NB4 cells into myeloid; the ATRA stimulated ERK activity in this process; ERK inhibitor PD98059 could partially block ATRA effect, specific inhibitor of p38MAPK, SB203580, combined with ATRA also could partially block the effects of ATRA on inhibition of NB4 growth and induction of differentiation. It is concluded that the ATRA stimulates ERK and p38MAPK pathway in the process inducing differentiation of NB4 cells, the ERK and P38MAPK may be necessary for the ATRA-induced differentiation in NB4 cells.

Effect of bufalin-inducing apoptosis on Bcl-2 and PKC in HL-60 cells / 中国实验血液学杂志

Previous study revealed that bufalin can inhibit proliferation, and induce apoptosis in some human cancer cell lines. However, the mechanism of its anticancer effect has not been fully understood. The present study was designed to investigate the effects of bufalin-induced apoptosis on Bcl-2 and PKC in human leukemic HL-60 cells. The cell viability was determined by trypan blue dye exclusion. The apoptosis was detected by morphology, flow cytometry and DNA agarose gel electrophoresis. The expressions of Bcl-2 and PKC were analyzed by Western blot, and activity of PKC was assayed by [gamma-(32)P] isotope incorporation method. The results showed as follows: (1) proliferation of HL-60 cells was inhibited by bufalin and the IC(50) at 24, 48, 72 hours were (25.8 +/- 2.1), (8.0 +/- 1.2) and (2.3 +/- 0.3) nmol/L, respectively. (2) apoptosis of HL-60 cells was induced when the cells were treated with bufalin at concentration of 50 nmol/L for 24 hours. (3) compared with control, treatment with bufalin at concentration of 50 nmol/L for 6 - 24 hours resulted in downregulation of protein expression, decrease of phosphorylation, and cleavage of Bcl-2, simultaneously. (4) the activity of total PKC was unchanged when HL-60 cells were exposed to 1 - 100 nmol/L bufalin for 30 minutes, but PKCbetaII underwent translocation from cytosol to membrane. It is concluded that apoptosis induced by bufalin is associated with downregulation of protein expression, dephosphorylation, and cleavage of Bcl-2 in HL-60 cells.

P38 mitogen-activated protein kinase mediates glucocorticoid receptor function induced by dexamethasone in acute lymphoblastic leukemia cells / 中华儿科杂志

<p><b>OBJECTIVE</b>Glucocorticoid (GC) has occupied a central role in the treatment of acute lymphoblastic leukemia due to its ability to induce apoptosis in neoplastic lymphoid cells. Glucocorticoid resistance is present among 20% initial acute lymphoblastic leukemia, even 80% refractory acute lymphoblastic leukemia. Glucocorticoid resistance has been an important determinant of clinical outcome. Glucocorticoid depends on glucocorticoid receptor (GR) to induce apoptosis. Glucocorticoid receptor, a number of nuclear hormone receptor superfamily, is mediated by many signal transduction systems. The mitogen-activated protein kinases (MAPK) superfamily of serine/threonine kinases has emerged as an important component of cellular signal transduction. Four MAP kinase families, ERK, p38 MAP kinase, JNK, and ERK5, have been well characterized. p38 MAPK usually plays a role in regulating apoptosis, cell cycle arrest and cytokines production, et al. In steroid resistance patients, IL-2 combined with IL-4 can decrease glucocorticoid receptor ligand-binding affinity via p38MAPK. In human alveolar epithelial A549 cells, dexamethasone could inhibit the activation of p38MAPK. It is unclear that the effect of p38MAPK on glucocorticoid receptor function induced by dexamethasone in CEM cells. This study aimed to investigate effect of p38 mitogen-activated protein kinase on glucocorticoid receptor function induced by dexamethasone in CEM cells.</p><p><b>METHODS</b>Cell viability was determined by trypan blue dye exclusion. Apoptosis was evaluated by morphology and flow cytometry. Glucocorticoid receptor protein and p-p38MAPK protein were analyzed by Western Blot.</p><p><b>RESULTS</b>When treatment with SB203580 and dexamethasone for 24 h to 72 h, the survival percentage was increased from 62.3%, 35.5% and 11.6% to 82.8%, 54.7% and 48.1%, respectively (P < 0.01). Co-treatment with SB203580 and dexamethasone resulted in the decrease of apoptotic percentage from 26.2% to 7.1% for 36 h (P < 0.01). p38 MAPK activation was apparent at 15 min, peaked at 1 h after dexamethasone treatment, and was sustained for 6 h. The phosphorylation was still observed at 48 h. Treatment with dexamethasone at 5 micromol/L for 12, 24, 36 and 48 h resulted in increase of GR(alpha) protein to 117%, 121%, 122% and 125% respectively. Unbinding to dexamethasone, GR(alpha) is in the cytoplasm. Nuclear-to-cytoplasmic ratio of GR(alpha) is 0.27. Treatment with dexamethasone at the same concentration and time resulted in the nuclear-to-cytoplasmic ratio increase to 0.48, 0.59, 0.95, 2.16 and 4.08 respectively. Combined treatment with SB203580 and dexamethasone resulted in the nuclear-to-cytoplasmic ratio decrease from 4.08 to 0.43 for 48 h (P < 0.05). The total GR(alpha) protein was unaffected.</p><p><b>CONCLUSIONS</b>Expression of GR(alpha) protein is upregulated and translocated into nucleus. p38MAPK enhances GR(alpha) protein translocation into nucleus.</p>

Expression of survivin gene in apoptosis induced by dexamethasone in CEM cells / 中国当代儿科杂志

<p><b>OBJECTIVE</b>The precise mechanism of glucocorticoid-induced apoptosis has not yet been elucidated. Survivin, a member of the inhibitors of apoptosis protein family, correlates with inhibition of apoptosis, proliferation, angiogenesis and multiple drugs resistance. This study aimed to investigate the variation of the survivin gene expression in apoptosis induced by dexamethasone (Dex) in the human T-lineage acute lymphoblastic leukemia (ALL) cell line, CEM-WT cells.</p><p><b>METHODS</b>The logarithmically growing CEM cells cultured in vitro (cell density 2 x10(5)/mL) were exposed to 0.1, 0.5, 1, 5, and 10 microM Dex, then were collected 24, 48 and 72 hrs later. Untreated CEM cells were used as Controls. The cell viability was determined by trypan blue dye exclusion. Apoptosis was evaluated by morphology and flow cytometry. Survivin protein and gene were analyzed by Western Blot and RT-PCR.</p><p><b>RESULTS</b>CEM cells growth was obviously inhibited by 0.1, 0.5, 1, 5, and 10 microM Dex from 48 hrs. The inhibition effect was dose- and time-dependent. CEM cells treated with Dex (> or = 5 microM) exhibited typical apoptotic features. The apoptosis increased after 5 microM Dex treatment in a time-dependent manner, with the apoptosis percentage increasing from 14.9% (12 hrs) to 46.2% (48 hrs). Compared with that of the Control group, the expression of survivin protein was down-regulated, with the expression rate of 54.6%, 45.5%, 15.8% and 9.7% respectively at 12, 24, 48 and 72 hrs after 5 microM Dex treatment. 5 microM Dex treatment also resulted in a decrease of survivin mRNA expression. The survivin mRNA expression was 76.4%, 67.3%, 55.0%, 49.9%, 38.3% and 18.3% of the Control respectively at 6, 12, 24, 48 and 72 hrs after Dex treatment.</p><p><b>CONCLUSIONS</b>Apoptosis induced by Dex in CEM cells is associated with downregulation of the survivin expression.</p>

Downregulation of Bcl-2 and survivin expression and release of Smac/DIABLO involved in bufalin-induced HL-60 cell apoptosis / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the expression of mitochondria-mediated apoptosis pathway related genes bcl-2, Bax, survivin and Smac/DIABLO in bufalin induced HL-60 cell apoptosis.</p><p><b>METHODS</b>Cell viability was determined by trypan blue exclusion, apoptosis by morphology and flow cytometry, expressions of Bcl-2, Bax, Survivin, Smac/DIABLO and caspase-3 protein by Western blot, and expression of survivin mRNA by RT-PCR.</p><p><b>RESULTS</b>Proliferation of HL-60 cells was inhibited by bufalin and the IC(50) at 24, 48 and 72 h were 25.8, 8.0 and 2.1 nmol/L, respectively. Apoptosis was induced when the cells were treated with bufalin at concentrations of 50.0 nmol/L and higher. Compared with the control, HL-60 cells treated with bufalin at 50.0 nmol/L for 6, 12, 24 and 48 h showed decrease of Bcl-2 protein expression to 88.6%, 53.3%, 19.2% and 9.5%, Bcl-2/Bax ratio (control 2.0) to 1.7, 1.1, 0.4 and 0.2, Survivin protein expression to 75.2%, 54.8%, 37.5% and 20.3%, and survivin mRNA to 85.7%, 39.4%, 12.5%and 0%, respectively. The expression of Smac/DIABLO protein was downregulated to 77.5% (12 h), 21.2% (24 h) and 15.3% (48 h) in mitochondrial fraction and upregulated to 1.4-(12h), 2.0-(24 h) and 3.5- folds (48 h) in cytosolic fraction, respectively. The active subunits of caspase-3 were displayed after treatment for 8 h till 48 h.</p><p><b>CONCLUSION</b>Apoptosis induced by bufalin is related to downregulation of expressions of bcl-2 and survivin, decrease of Bcl-2/Bax ratio, mitochondrial release of Smac/DIABLO, and activation of caspase-3. The mitochondria-mediated apoptotic pathway may be involved in the apoptosis induced by bufalin in HL-60 cells.</p>

Leukocytosis and retinoic acid syndrome in patients with acute promyelocytic leukemia treated with arsenic trioxide / 中国医学科学杂志(英文版)

<p><b>OBJECTIVE</b>To study the incidence of leukocytosis and retinoic acid (RA) syndrome in newly diagnosed and relapsed acute promyelocytic leukemia (APL) patients treated with arsenic trioxide (ATO).</p><p><b>METHODS</b>Thirty patients with newly diagnosed or relapsed APL received ATO for remission induction at the dose of 10 mg/d. RA syndrome was defined when patient was with one or more of the following signs or symptoms: fever, dyspnea, serous cavity effusion, muscular pain, pulmonary infiltration, weight gain, or pulmonary infiltration on chest X-ray.</p><p><b>RESULTS</b>Twenty-three (77% ) patients achieved complete remission, mean time to remission was 37.1 days. Leukocytosis was observed in 14 (47%) patients, mean time to leukocytosis was 12.7 days, median baseline leukocyte count for patients with leukocytosis was 3.1 x 10(9)/L, which was higher than that for patients who did not develop leukocytosis (2.6 x 10(9)/L, z = -2.635, P = 0.008). No other cytotoxic therapy was administered, and the leukocytosis resolved in all cases. The RA syndrome was observed in 9 (30%) patients, mean time to diagnose of RA syndrome was 13.9 days, median baseline leukocyte count for patients with RA syndrome was 3.6 x 10(9)/L, which was higher than that for patients who did not develop RA syndrome (2.6 x 10(9)/L, z = -1.909, P = 0.046). No patient died of RA syndrome.</p><p><b>CONCLUSION</b>Leukocytosis and RA syndrome are associated with ATO and baseline leukocyte count respectively, and there is distinct link between leukocytosis and RA syndrome.</p>

Effect of LY294002 and its combination with chemotherapy drugs on the proliferation of human leukemia K562 cell line and its possible mechanism / 中国癌症杂志

Background and purpose:Remarkable advances have been made in cancer chemotherapy by developing new anticancer drugs and therapy strategies.However,multidrug resistance in human cancers remains a major clinical challenge for cancer treatment.Attempts in several clinical studies to reverse multidrug resistance protein (MDR) by using MDR modulators have not yet generated promising results.Our aim was to explored the mechanism of reversal of multidrug resistance in human leukemia K562 cells by PI3-K inhibitor.Methods:Trypanblue dye exclusion method was used to observe the drug sensitivity and the effect of LY294002 on the drug resistance.Western blot to analyze P-gp and p-Akt phenotypes,and flow cytometer was used to measure the intracellular drug accumulation. Results:K562/D induced by DNR was cross resistant to DNR,ADR,VCR and VP16 (Resistance Index:65,52,134 and 50 respectively).DNR induced over-expressions of P-gp and p-Akt in K562/D cells;LY294002 increased the intracellular drug accumulation,and then reversed the drug resistance to DNR,ADR,VCR and VPI6 in K562/D cells(Resistance Index:23,21,63 and 29 respectively),but not in the sensitive cells (K562/S).Conclusion:The multidrug resistance of K562/D cells can be induced by DNR which is related to the P-gp and p-Akt over-expressions, and LY294002 can reverse multidrug resistance in human leukemia cells in vitro via inhibits PI3-K/Akt pathway.