Down-modulation of heat shock protein 70 and up-modulation of Caspase-3 during schisandrin B-induced apoptosis in human hepatoma SMMC-7721 cells

AIM: To investigate the effect of schisandrin B (Sch B) on proliferation and apoptosis of human hepatoma SMMC-7721 cells in vitro and regulation of Hsp70 and Caspases-3, 7, 9 expression by Sch B. METHODS: Human hepatoma cell line SMMC-7721 was cultured and treated with Sch B at various concentrations. Growth suppression was detected with MTT colorimetric assay. Cell apoptosis was confirmed by DNA ladder detection and flow cytometric analysis. The expression of Hsp70, Caspases-3, 7, 9 were analyzed by Western blot analysis. RESULTS: Sch B inhibited the growth of hepatoma SMMC-7721 cells in a dose-dependent manner, leading to a 50% decrease in cell number (LC50) value of 23.50 mg/L. Treatment with Sch B resulted in degradation of chromosomal DNA into small internucleosomal fragments, evidenced by the formation of a 180-200 bp DNA ladder on agarose gels. FCM analysis showed the peak areas of subdiploid at the increased concentration of Sch B. The results of Western bolt analysis showed that Hsp70 was down-regulated and Caspase-3 was up-regulated, while the activity of Caspases-7, -9 had no significant change. CONCLUSION: Sch B is able to inhibit the proliferation of human hepatoma SMMC-7721 cells and induce apoptosis, which goes through Caspase-3-dependent and Caspase-9-independent pathway accompanied with the down-regulation of Hsp70 protein expression at an early event.     

Hepatosplenic γδ T-cell lymphoma

AIM: To investigate the clinicopathologic characteristics, immunophenotype and TCR gene rearrangements of hepatosplenic T-cell lymphoma in eight Chinese patients. METHODS: Eight Chinese patients with hepatosplenic 76 T-cell lymphomas were studied. Hematoxylin-eosin-stained slides and clinical histories were reviewed. We also carried out immunohistochemical staining for CD3, CD4, CD8, CD20, CD43, CD56, CD79a, UCHL-1, and TCR γδ. Rearrangements of TCR gamma and delta chain genes were also studied. RESULTS: The spleens were enlarged and the cut surfaces were homogeneous and red-purple in color without identifiable gross lesions or enlarged hilar lymph nodes. Histologically, lymphoma cells infiltrated the cords of Billroth and often packed the sinuses. Liver biopsy showed lymphoma cell infiltrations in the sinusoids, and three cases showed involvements of the portal tracts. Immunohistochemically lymphoma cells were positive for CD3, CD43, and CD56 in all cases. Four of eight cases were positive for CD8, and all cases were negative for CD4 (6/6). Monoclonal rearrangements of TCR y gene were demonstrated by PCR analysis in five out of the eight cases. TCR δ gene rearrangements were detected in six out of the eight cases, which demonstrated single bands on PAGE gel, and the amplification products in two cases were confirmed by sequencing. CONCLUSION: The clinicopathology of hepatosplenic γδ T-cell lymphoma in Chinese patients is similar to what was previously reported except that the splenomegaly is not so massive, and CD8 is positive.     

阿托伐他汀降低急性冠脉综合征患者外周血CD_(14)~+单核细胞表面TLR4的表达

目的 Toll 样受体4(TLR4)的激活与动脉粥样斑块的进展及斑块的不稳定导致临床并发症的发生有关。他汀类调脂药物临床获益可能与其调脂以外的作用尤其是抗炎作用有关,但抗炎作用的确切机制目前还不清楚。推测其抗炎作用可能是通过抑制 TLR4炎症信号通路起作用的。方法入选者为健康志愿者(n=22)及2006-07-2007-09在门诊和住院患者共121例[稳定性心绞痛(SAP)患者17例,急性冠脉综合征(ACS)患者82例],入院即刻抽取外周静脉血;将其中的41例 ACS 患者随机分为两组:在常规抗心绞痛治疗的基础上服用阿托伐他汀10 mg 组(20例)及阿托伐他汀40 mg 组(21例),治疗1月后抽取外周静脉血。观察指标为血脂、高敏 C 反应蛋白(hsCRP)、外周血 CD_(14)~+单核细胞表面 TLR4的表达,单核细胞表面 TLR4的表达采用流式细胞仪方法测定。结果 ACS 患者血 hsCRP 及外周血 CD_(14)~+单核细胞表面 TLR4的表达明显高于 SAP 患者(P<0.05)及正常对照组(P<0.05),hsCRP 与 TLR4的相关性分析结果是两者轻度相关(r=0.261,P=0.002)...     

Anti-VEGF agents confer survival advantages to tumor-bearing mice by improving cancer-associated systemic syndrome

The underlying mechanism by which anti-VEGF agents prolong cancer patient survival is poorly understood. We show that in a mouse tumor model, VEGF systemically impairs functions of multiple organs including those in the hematopoietic and endocrine systems, leading to early death. Anti-VEGF antibody, bevacizumab, and anti-VEGF receptor 2 (VEGFR-2), but not anti-VEGFR-1, reversed VEGF-induced cancer-associated systemic syndrome (CASS) and prevented death in tumor-bearing mice. Surprisingly, VEGFR2 blockage improved survival by rescuing mice from CASS without significantly compromising tumor growth, suggesting that “off-tumor” VEGF targets are more sensitive than the tumor vasculature to anti-VEGF drugs. Similarly, VEGF-induced CASS occurred in a spontaneous breast cancer mouse model overexpressing neu. Clinically, VEGF expression and CASS severity positively correlated in various human cancers. These findings define novel therapeutic targets of anti-VEGF agents and provide mechanistic insights into the action of this new class of clinically available anti-VEGF cancer drugs.     

Human endostatin gene transfer, either naked or with liposome, has the same inhibitory effect on growth of mouse liver tumor cells in vivo

AIM: To explore a safe and efficient strategy of tumor therapy using anti-angiogenetic agents. METHODS: Endostatin gene with a signal sequence of human IgGgamma chain was amplified by PCR and cloned into pVAX1 plasmid which was the only vector authorized by FDA in clinical trial to construct a recombinant plasmid named as pVAX-sEN. The recombinant plasmid was detected with EcoRI/KpnI and DNA sequencing. BALB/c mice bearing hepatocarcinoma cell line H22 were treated with naked pVAX-sEN or liposome-DNA complex in which the dose of DNA and the ratio of DNA and liposome were different from each other. To compare the efficiency of gene transfection, expression of endostatin at the treated tumor site was assayed with ELISA. To investigate the effect of pVAX1-sEN on hepatocellular carcinoma, pVAX-sEN either naked or in liposome-DNA complex was injected into BALB/c mice bearing H22, then the diameter of tumors was measured, microvessel density was detected by immunohistochemistry, endostatin expression in vivo was assayed at different time points. RESULTS: DNA sequencing showed the endostatin gene with the signal peptide was correctly cloned. In situ gene expression assay indicated that both the ratio of DNA and liposome and the dose of DNA could affect the gene transfection efficiency. Interestingly, naked pVAX-sEN had a similar in situ endostatin expression to pVAX-sEN with liposome. Animal experiments showed that pVAX-sEN together with pVAX-sEN-liposome complex could efficiently suppress the growth of mouse hepatoma cells. CONCLUSION: Naked endostatin plasmid intratumoral injection can get a similar gene transfection efficiency to liposome-DNA complex when used in situ.     

Analysis of spontaneous,gamma ray- and ethylnitrosourea-induced hprt mutants in HL-60 cells with multiplex PCR

AIM: To explore the molecular spectra and mechanism of human hypoxanthine guanine phosphoribosyl transferase (hprt) gene mutation induced by ethyluitrosourea (ENU) and 60Co γ-rays.METHODS: Independent human promyelocytic leukemia cells (HL-60) mutants at the hprt locus were isolated from untreated, ethyluitrosourea (ENU) and 60Co γ-ray-exposed cells, respectively, and verified by two-way screening. The genetic changes underlying the mutation were determined by multiplex polymerase chain reaction (PCR) amplification and electrophoresis technique.RESULTS: With dosage increased, survival rate of plated cell reduced (in the group with dosage of ENU with 100-200μg/ml, P＜0.01; in the group with dosage of 60Co γ-ray with 2-4 Gy, P＜0.05) and mutational frequency increased (in the group of ENU 12.5-200.0 μg/ml, P＜0.05; in the group of 60Co γ-ray with 1-4 Gy, P＜0.05) significantly. In the 13spontaneous mutants analyzed, 92.3 % of mutant clones did not show any change in number or size of exon, a single exon was lost in 7.7 %, and no evidence indicated total gene deletion occurred in nine hprt exons. However, deletions were found in 79.7 % of ENU-induced mutations (62.5-89.4 %,P＜0.01) and in 61.7 % of gamma-ray-induced mutations (28.6-76.5 %, P＜0.01). There were deletion mutations in all 9 exons of hprt gene and the most of induced mutations were chain deletion with multiplex exons (97.9 % in gammaray-induced mutants, 88.1% in ENU-induced mutants).CONCLUSION: The spectra of spontaneous mutations differs completely from that induced by EUN or 60Co γ-ray.Although both ENU and γ-ray can cause destruction of genetic structure, mechanism of mutagenesis between them may be different.     

Effects of splenectomy in patients with cirrhosis undergoing hepatic resection for hepatocellular carcinoma

AIM: To study the effects of splenectomy in patients with cirrhosis undergoing hepatic resection for hepatocellular circinoma.METHODS: Twenty-six patients with HCC associated with cirrhosis were divided into hepatectomy with splenectomy group (splenectomy group, n=11) and hepatectomy without splenectomy group (non-splenectomy group, n=15). T lymphocyte subsets such as CD4, CD8, CD4/CD8, helper T (Th) lymphocyte cytokines such as interferon γ(IFN-γ),interleukin 2(IL-2), interleukin 10(IL-10) and white blood cell (WBC), platelet (PLT), total bilirubin (T-Bil) were measured and used as parameters to evaluate the effects of splenectomy.RESULTS: There was no significant difference in CD4, CD8,CD4/CD8, IL2, IFN-γ, IL10, WBC, PLT, T-Bil levels between two groups before surgery. Two months after operation,the levels of CD4 (41.2 %±4.2 % vs 34.7 %±3.8 %), CD4/CD8 (1.7±0.2 vs 1.0±0.2), IFN-γ (102.3±15.9 pg/ml vs 86.5±14.8 pg/ml), IL-2(97.2±15.6 pg/ml vS77.6±14.5 pg/ml) were increased and those of CD8 (25.6±3.9 vs32.8 %±4.1%),IL-10 (56.9±10.4 pg/ml vs 72.6±15.3 pg/ml) were decreased in splenectomy groups as compared with those in nonsplenectomy group (P<0.05). WBC and PLT counts in the splenectomy group were 8.9±1.6×109 and 310±32×109,respectively, which were significantly higher than those in non-splenectomy group (3.7±1.4×109 and 104±41×109) respectively on the 14th post-operative day. T-Bil concentration in the splenectomy group (24±7 μmol/L) was significantly lower than that in the non-splenectomy group (37±13 μmol/L) on the 7th post-operative day (P<0.05).CONCLUSION: Splenectomy combined with hepatectomy for HCC associated with cirrhosis is helpful for the recovery of T-lymphocyte subsets and the maintenance of Th:L/Th2 cytokine balance.     

p16INK4A and p15INK4B gene deletions in primary leukemias

The 9p21 locus has been deleted at a high frequency in a wide variety of tumors. Recently, two genes, p16INK4A and p15INK4B (also called MTS1 and MTS2), have been localized in close proximity at the 9p21 locus, encoding cyclin-dependent kinases 4/6 inhibitors of relative molecular mass 16 kD and 15 kD, respectively and also found to be deleted at a high frequency in tumor cell lines. We analyzed p16INK4A and p15INK4B genes in 178 cases of primary leukemias including 81 cases of chronic lymphocytic leukemia (CLL), seven of hairy cell leukemia (HCL), seven of chronic myelogenous leukemia (CML), 43 of acute myelogenous leukemia (AML), 27 of acute lymphoblastic leukemia (ALL), and 13 of myelodysplastic syndrome (MDS) by Southern blot analyses. The ALL cases showed a relatively high frequency of homozygous deletions (22%, 6 of 27) at the p16INK4A gene locus. Interestingly, of the six cases with p16INK4A homozygous deletions, only three showed homozygous deletions at the p15INK4B gene. In 81 CLL patients, we detected one homozygous and five heterozygous deletions at both the p16INK4A and p15INK4B genes and two heterozygous deletions at the p16INK4A gene alone. Deletion of these two genes in AML cases is relatively low (9%). We did not detect deletions in any of the MDS, HCL, and CML cases examined. Sequence analyses of p16INK4A gene of six CLL cases with heterozygous deletion at this locus showed a 27-bp deletion at the splice acceptor site of intron 1 in one case and changes in the coding sequence in three other cases. The data presented in this report showed that (1) p16INK4A and p15INK4B genes are preferentially deleted homozygously in ALL and heterozygously in CLL cases with frequent mutation in the second allele, and (2) p16INK4A gene appears to be more frequently deleted than p15INK4B gene.     

“快－慢”型室上性心律失常的射频导管消融治疗及随访

目的：探讨射频导管消融在治疗“快—慢”型室上性心律失常中的作用。方法：用射频导管消融的方法治疗了４例“快—慢”型室上性心律失常病人。结果：１９９１年１月至１９９６年１月间，对２４１例次各种快速性心律失常进行了射频导管消融治疗。其中４例为“快—慢”型室上性心律失常，２例为反复发作性房室结折返性心动过速（ＡＶＮＲＴ），终止时有长时间的心脏停搏并引起晕厥，原准备安装永久性心脏起搏器，ＡＶＮＲＴ根治后，由其引起的症状不复存在，电生理检查窦房结功能正常，故未安装起搏器；另２例均已植入永久性心脏起搏器，１例频繁发作快速心室率心房颤动并经常引起急性心功能不全，１例反复发作ＡＶＮＲＴ、心房扑动和心房颤动且有明显的症状，射频导管消融治疗后症状均消失，射频导管消融术对起搏器的功能无影响。３例平均随访３１±２个月，１例随访２个月未见并发症和临床症状复发。结论：射频导管消融法治疗“快—慢”型室上性心律失常具有重要的临床价值。