Cellular and Biochemical Mechanisms of the Resistance of Human Cancer Cells to a New Anticancer Ribo-nucleoside, TAS-106

We have established variants of DLD-1 human colon carcinoma and HT-1080 human fibrosarcoma cells resistant to the new anticancer ribo -nucleosides, 1-(3- C -ethynyl-β-D-ribo-pentofuranosyl)-cytosine (ECyd, TAS-106) and 1-(3- C -ethynyl-p-D-ribo-pentofuranosyl)uracil (EUrd). Both variants were shown to have decreased (3- to 24-fold decrease) uridine-cytidine kinase (UCK) activity, and exhibited cross-resistance to EUrd and TAS-106. Based on the IC₅₀ values determined by chemosensitivity testing, a 41- to 1102-fold resistance to TAS-106 was observed in the resistant cells. TAS-106 concentration-dependently inhibited RNA synthesis, while its effect on DNA synthesis was negligible. The degree of resistance (14- to 3628-fold resistance) calculated from the inhibition of RNA synthesis tended to be close to the degree of chemoresistance of tested cells to TAS-106. The experiments on the intracellular metabolism of TAS-106 in the parental cells revealed a rapid phosphorylation to its nucleotides, particularly the triphosphate (ECTP), its major active metabolite. The amount of TAS-106 transported into the resistant cells was markedly reduced and the intracellular level of ECTP was decreased from 1/19 to below the limit of detection; however, the unmetabolized TAS-106 as a percentage of the total metabolite level was high as compared with the parental cells. The ratio of the intracellular level of ECTP between parental and resistant cells tended to approximate to the degree of resistance calculated from the inhibitory effect on RNA synthesis. These results indicate that the TAS-106 sensitivity of cells is correlated with the intracellular accumulation of ECTP, which may be affected by both the cellular membrane transport mechanism and UCK activity.     

Aurora-A基因在肺鳞癌组织中的表达

目的 探讨Aurora-A基因在肺鳞癌组织中的表达.方法 采用免疫组化方法检测Aurora-A基因蛋白在肺鳞癌及其癌旁组织中的表达水平.结果 48例肺鳞癌中32例肿瘤组织Aurora-A基因表达率（66.67%）较其对应癌旁组织高,且表达水平与肺鳞癌的转移和临床分期相关.结论 Aurora-A基因在肺鳞癌组织中呈过表达,因此,Aurora-A基因有望成为新的肺癌分子治疗靶点和肿瘤标记物.     

p120-catenin和Rac1的协同表达与非小细胞肺癌恶性程度相关性的研究

背景与目的 在肺癌组织中,p120-catenin(p120ctn)与small GTP酶家族中的主要成员Rac1的表达是否具有相关性,至今尚不清楚.方法 本研究应用S-P免疫组化、Western Blot、RT-PCR方法观察138例肺癌标本及2种具有不同侵袭转移能力的同源肺癌细胞系中p120ctn和Rac1的表达.结果 在肺癌组织中p120ctn的蛋白和mRNA表达水平均明显低于正常肺组织,而Rac1在肺癌组织中的表达明显高于正常肺组织,p120ctn的异常表达与Rac1的过表达有较好的相关性(Correlation coefficient=0.720,P＜0.001),并与肺癌的分化(P=0.022),分期(P=0.010)和淋巴结转移(P=0.009)相关.同时,在肺癌细胞系中我们还观察到p120ctn表达下降和Rac1的过表达现象在具有高转移能力的BE1细胞系中尤为突出.结论 肺癌中p120ctn的异常表达与Rac1的过表达有关,并与肺癌的恶性程度相关.     

Expression of C-terminal src Kinase in Human Colorectal Cancer Cell Lines

C-terminal src kinase (CSK) is a cytoplasmic protein which decreasesactivities of the Src family protein-tyrosine kinases. We produceda polyclonal antibody specific for CSK and analyzed the expressionof CSK by immunoblotting in two human colorectal normal celllines and eighteen cancer cell lines. CSK was detected in boththe two normal and all the eighteen cancer cell lines. The expressionof CSK obtained from human colorectal cancer cell lines wasgreater than that from human colorectal normal cell lines inmost cases. The elevated expression of CSK in human colorectalcancer cell lines appeared to correspond to the elevated p60^c-src(c-Src) and p62^c-yes (c-Yes) protein-tyrosine kinase activitiesfound in other studies. Thus, CSK may not have an anti-oncogenicrole to play through the negative regulation of Src family kinasesin colorectal carcinogenesis     

miR-106a和miR-24-1在结肠癌中的表达及意义

 目的
检测结肠癌与癌旁组织miR-106a和miR-24-1的表达差异及其与c-myc的表达是否具有关联性。方法选临床病理诊断明确的结肠癌患者,手术后取癌组织标本提取RNA,RT-PCR检测原癌基因c-myc在结肠癌组织与癌旁组织中的表达。TaqMan荧光定量PCR检测结肠癌与癌旁组织miR-106a和miR-24-1的表达差异。结果c-myc表达阳性的癌组织表达miR-106a要明显高于癌旁组织,其差异具有统计学意义（P<0.05）;c-myc表达阴性的癌组织表达miR-106a要高于癌旁组织（P<0.05）,结肠癌组织与癌旁组织miR-24-1的表达无差异。结论结肠癌组织c-myc阳性组和c-myc阴性组表达miR-106a均高于癌旁组织,miR-106a有可能作为结肠癌的筛查目标之一;结肠癌组织c-myc阳性组和c-myc阴性组表达miR-24-1与癌旁组织无差异,还需更多研究证实其与结肠癌的关系。     

Caffeine Enhancement of the Effect of Anticancer Agents on Human Sarcoma Cells

It is necessary to find modifiers which enhance the effects of known anticancer agents in order to improve both survival rate and local curability of patients with high-grade sarcomas. In this study, the effect of anticancer agents combined with caffeine was examined on cultured sarcoma cells and fresh human sarcoma specimens, utilizing the human tumor clonogenic assay technique. The combination of cisplatin and caffeine showed a synergistic inhibition of the growth of two strains of cultured sarcoma cells tested, and 14 of 18 fresh human sarcoma specimens (77.8%). This synergistic effect of caffeine was also observed with cyclophosphamide (44.8%), mitomycin C (44.8%) and adriamycin (27.8%). The combination of vincristine or methotrexate with caffeine, however, did not exhibit a synergistic effect. Caffeine, therefore, enhanced the effect of four cytotoxic DNA damaging agents. No antag- onistic effects were seen in our series. This study suggests that caffeine may be useful in enhancing the tumoricidal effect of anticancer drugs, especially DNA-damaging agents, and possibly may aid in overcoming natural drug resistance.     

Deletion Mutants of Human Deoxycytidine Kinase mRNA in Cells Resistant to Antitumor Cytosine Nucleosides

We studied mutational events in deoxycytidine (dCyd) kinase mRNA expression, focusing on aberrant dCyd kinase mRNA, which has been frequently observed in established cell lines resistant to antitumor dCyd nucleoside analogues such as 1-β-D-arabinofuranosyl cytosine (Ara-C), gemcita-bine (dFdC) and 2'-C-cyano-2'-deoxy-l-β-D-arabinofuranosylcytosine (CNDAC). We describe here the expression of aberrant dCyd kinase mRNAs identified as splicing mutants. These mutants included deletions of the fifth exon in CNDAC-resistant cells (originating from HT-1080 cells), of the third exon in Ara-C-resistant cells (originating from SK-MEL-28 cells) and of the fourth exon in 2'-deoxy-2'-methylidenecytidine (DMDC)-resistant cells (originating from SK-MEL-28 cells). Various nucleoside-resistant cells originating from the same parental HT-1080 cells were established. The resulting cells expressed the same mRNA with deletion of the fifth exon, and the location of splicing was independent of the type of nucleosides used for the establishment of resistant cells. The deletion of the fifth exon in dCyd kinase seems to be a target for acquisition of resistance to antitumor cytosine nucleosides. However, distinct mutations in the dCyd kinase gene seem to be associated with acquisition of resistance to different antitumor cytosine nucleosides.     

Sensitivity of Gastric Cancer Cells to Chemotherapy Drugs in Elderly Patients and Its Correlation with Cyclooxygenase-2 Expression

Objective: To explore the sensitivity of gastric cancer cells to chemotherapy drugs in elderly patients andits correlation with cyclooxygenase-2 (COX-2) expression in cancer tissue. Materials and Methods: Forty-threeelderly patients with gastric cancer (observation group) and 31 young patients with gastrointestinal tumors(control group) who were all diagnosed by pathology and underwent surgery in the 89th Hospital of ChinesePeople’s Liberation Army were selected. Drug sensitivity testing of tumor cells in primary culture was carriedout in both groups using a methyl thiazolyl tetrazolium (MTT) method, and the expression of COX-2 and thefactors related to multi-drug resistance (MDR) in cancer tissue were assessed by immunohistochemistry. Results:The inhibition rates (IR) of vincristine (VCR), 5-fluorouracil (5-FU), oxaliplatin (L-OHP), mitomycin (MMC)and epirubicin (eADM) on tumor cells in the observation group were dramatically lower than in the controlgroup, with statistical significance (P<0.05 or P<0.01). The positive rates of COX-2, glutathione s-transferase-π(GST-π) and P glycoprotein (P-gp) expression in cancer tissue in the observation group were all higher than incontrol group (P<0.05), while that of DNA topoisomerase IIα (TopoIIα) expression lower than in the control group(P<0.01). In the observation group, COX-2 expression in cancer tissue had a significantly-positive correlationwith GST-π and P-gp (r=0.855, P=0.000; r=0.240, P=0.026), but a negative correlation with TopoIIα (r=−0.328,P=0.002). In the control group, COX-2 expression in cancer tissue was only correlated with P-gp positively(r=0.320, P=0.011). Bivariate correlation analysis displayed that COX-2 expression in cancer tissue in theobservation group had a significantly-negative correlation with the IRs of 5-FU, L-OHP, paclitaxel (PTX) andeADM in tumor cells (r=−0.723, P=0.000; r=−0.570, P=0.000; r=−0.919, P=0.000; r=−0.781, P=0.000), but withhydroxycamptothecine (HCPT), VCR and 5-FU in the control group (r=−0.915, P=0.000; r=−0.890, P=0.000;r=−0.949, P=0.000). Conclusions: Gastric cancer cells in elderly patients feature stronger MDR, which may berelated to high COX-2 expression.     

Cytochrome 4Z1 Expression Is Correlated with Poor Prognosis in Patients with Cervical Cancer

Background: cervical cancer is one of the most common malignancies in women worldwide and its management remains challenging and complex. As Cytochrome4Z1 (CYP4Z1) is overexpressed in many tumours, its expression in cervical cancer is unknown. Therefore, the present study aimed to evaluate CYP4Z1 expression in cervical cancers. Methods: CYP4Z1 expression was immunohistochemically assessed in 100 cases of cervical cancers along with ten normal cervix tissues, and the enzyme’s relationship to several clinicopathological features and survival was explored. Results: CYP4Z1 was strongly expressed in 55% of cervical cancer patients. Normal cervix samples were negative for CYP4Z1 expression. Importantly, this expression was significantly found in patients with the late stage of the disease, lymph node metastasis, and high tumour invasion (p < 0.05). Interestingly, CYP4Z1 expression was significantly correlated with shorter survival times of cervical cancer patients. Univariate analysis showed that CYP4Z1 expression, tumour stage, lymph node metastasis, and tumour invasion were significantly correlated with patient survival (p < 0.05). The multivariate analysis revealed that only CYP4Z1 expression and tumour stage were significantly correlated with patient survival (p < 0.05). Conclusions: CYP4Z1 expression is associated with cervical cancer patients’ survival and may serve as an independent predictor of poor prognosis in cervical cancer patients.     

Enhancement of Cisplatin Sensitivity in High Mobility Group 2 cDNA-transfected Human Lung Cancer Cells

To elucidate the role of high mobility group 2 protein (HMG2) in cis -diamminedichloroplatinum (II) (cisplatin, CDDP) sensitivity, we constructed a human HMG2 -transfected human non-small cell lung cancer cell line, PC-14/HMG2. The HMG2 mRNA expression level was approximately twice those of parental PC-14 and mock-transfected PC-14/CMV. Gel mobility shift assay revealed a CDDP-treated DNA-protein complex in the nuclear extract of PC-14/HMG2, which was not found in the extracts of PC-14 and PC-14/CMV. This complex formation was subject to competition by CDDP-treated non-specific salmon sperm DNA, indicating that ectopic HMG2 recognizes CDDP-damaged DNA. PC-14/HMG2 showed more than 3-fold higher sensitivity to CDDP than PC-14 and PC-14/CMV. The intracellular platinum content of PC-14/HMG2 after exposure to 300 μM CDDP was 1.1 and 1.5 times that of PC-14 and PC-14/CMV, respectively. Cellular glutathione levels were not different in these cell lines. Repair of DNA interstrand cross-links determined by alkaline elution assay was decreased in PC-14/HMG2. These results suggest that HMG2 may enhance the CDDP sensitivity of cells by inhibiting repair of the DNA lesion induced by CDDP.     

Anticancer Effect of COX-2 Inhibitor DuP-697 Alone and in Combination with Tyrosine Kinase Inhibitor (E7080) on Colon Cancer Cell Lines

Colorectal cancer remains one of the most common types of cancer and a leading cause of cancer deathworldwide. In this study, we aimed to investigate effects of DuP-697, an irreversible selective inhibitor of COX-2 on colorectal cancer cells alone and in combination with a promising new multi-targeted kinase inhibitorE7080. The HT29 colorectal cancer cell line was used. Real time cell analysis (xCELLigence system) wasconducted to determine effects on colorectal cell proliferation, angiogenesis was assessed with a chorioallantoicmembrane model and apoptosis was determined with annexin V staining. We found that DuP-697 alone exertedantiproliferative, antiangiogenic and apoptotic effects on HT29 colorectal cancer cells. For the antiproliferativeeffect the half maximum inhibition concentration (IC50) was 4.28510-8 mol/L. Antiangiogenic scores were 1.2,0.8 and 0.5 for 100, 10 and 1 nmol/L DuP-697 concentrations, respectively. We detected apoptosis in 52% ofHT29 colorectal cancer cells after administration of 100 nmol/L DuP-697. Also in combination with the thyrosinekinase inhibitor E7080 strong antiproliferative, antiangiogenic and apoptotic effects on HT29 colorectal cancercells were observed. This study indicates that DuP-697 may be a promising agent in the treatment of colorectalcancer. Additionally the increased effects observed in the combination with thyrosine kinase inhibitor give thepossibility to use lower doses of DuP-697 and E7080 which can avoid and/or minimize side effects.     

常规化疗药物连续刺激对人肺腺癌细胞株表达erbB-2，p53和MDR1的影响

目的:分析常规化疗药物连续刺激对肿瘤细胞株表达erbB-2,p53和MDR1的影响,为联合应用生物治疗提供实验基础.方法:人肺癌细胞株经ADM,VP-16,DDP单药及联合用药连续刺激,每种药物分2个剂量梯度.应用流式细胞术分别检测erbB-2,p53和MDR1的阳性细胞数及平均荧光强度,以此推算出不同药物在不同浓度下连续2～3次作用后细胞株以上各蛋白表达的细胞数、平均表达量、总表达量,同时设对照组.结果:随着培养时间的延长各项检测指标其阳性细胞的百分数、平均荧光强度及总荧光强度均呈下降趋势.高剂量各组erbB-2和MDR1以上各项指标基本上均随刺激次数的增加呈下降趋势,而低剂量各组的检测指标则有升有降;p53的表达无一定规律,但第三次刺激后药物组的表达均高于对照组.结论:化疗药物连续刺激后,特别是小剂量化疗后,erbB-2,p53和MDR1的表达可不同程度增高,提示临床上针对这些分子对病人实 施生物治疗时应考虑化疗药物的影响.     

抗癌生物活性肽对BGC-823 细胞基因表达的影响*

目的:利用基因芯片技术分析一种新型抗癌生物制剂- 抗癌生物活性肽对人胃癌BGC-823 细胞的基因表达谱调控的影响,并对部分差异表达基因进行RT-PCR 检测,从而验证芯片的有效性。方法:订制表达谱芯片,分别将400 条和648 条人类基因的PCR 产物点样于特殊处理后的玻璃片上制备基因表达谱芯片。以加入不同浓度抗癌生物活性肽及处理不同时间的BGC-823 细胞为实验组,以生理盐水处理的BGC-823 细胞作为对照组,当确定最佳作用时间及抗癌生物活性肽有效作用浓度后,分别提取实验组与对照组细胞的总RNA,反转录成cDNA ,以两种荧光素Cy5 和Cy3 标记后作为探针,与制备好的表达谱芯片进行杂交,以生物信息学方法进行数据的处理和分析,并将其中的差异表达基因p15和HSP 701B 进行RT-PCR 的验证。结果:两张芯片上差异表达的基因共47个,其中上调27个,下调20个。这些基因按照功能分类涉及细胞周期、代谢酶、免疫相关、细胞凋亡、抑癌基因等类别,包括HSP 701B、HSP 75、HSP 90、磷脂酶D2（PLD 2）、凋亡抑制基因（IEX-1L）、人类杆状病毒IAP 重复序列3、E2 泛素耦联酶Ubch5c、人类缺氧诱导因子1 α 亚基、γ- 干扰素、TNF 诱导因子、人类黑色素瘤抗原p15基因等。同时RT-PCR 结果验证了芯片的检测的有效性。结论:抗癌生物活性肽具有调控人胃癌BGC-823 细胞基因表达的作用,其中对抑癌基因、热休克蛋白家族基因等有较明显的影响;基因芯片可以为阐明抗癌药物的作用机制提供有力的分子基础。      

PYK-2在PGE_2诱导大肠癌细胞侵袭转移中的作用

目的 研究富含脯氨酸的酪氨酸激酶 2(proline rich tyrosine kinase 2,PYK 2)在前列腺素E2(PGE2)诱导大肠癌SW480细胞侵袭转移中的作用。方法 实验分为A、B、C、D四组,分别为未处理组,PGE2组,PGE2+SC19220(EP1抑制剂)组,PGE2+BAPTA AM(胞内Ca2+螯合剂)组。通过 RT PCR检测SW480中PGE2四种EP(EP1,EP2,EP3,EP4)受体的表达,应用Western blotting检测SW480细胞中PYK 2蛋白的表达,应用Transwell实验观察各组SW480细胞侵袭转移能力的改变。结果SW480表达PGE2的三种EP受体,EP1,EP2和EP4,PGE2可促进EP1的表达;经PGE2作用后,10分钟内PYK 2磷酸化水平逐渐增加,0分钟、5分钟、10分钟检测结果相比差异均有统计学意义(P<0.05),30分钟与0分钟检测结果相比差异无统计学意义(P>0.05);C组、D组与B组相比PYK 2磷酸化水平明显下降(P<0.05),大肠癌细胞侵袭转移能力显著降低(P<0.05)。结论 PGE2可能通过Ca2+,EP1促进PYK 2的磷酸化,从而进一步诱导大肠癌细胞的侵袭转移过程。     

戊地昔布对人食管癌细胞凋亡及COX-2表达的影响

目的:探讨戊地昔布对人食管癌Eca109细胞系凋亡及环氧化酶-2表达的影响。方法:采用MTT法检测戊地昔布对人食管癌Eca109细胞生长的作用,流式细胞术检测细胞凋亡和细胞周期分布,电子显微镜进一步检测细胞凋亡,RT-PCR及流式细胞术检测COX-2mRNA及蛋白的表达。结果:戊地昔布(25～400μmol/L)依时间和浓度依赖性抑制人食管癌Eca109细胞的生长,作用72h后,对细胞生长的抑制率可达85.95%,凋亡率由(2.38±0.42)%增加到(48.46±0.73)%;50～400μmol/L时增殖指数和S期的细胞比例则明显降低,G0/G1期的细胞比例增加;同时,低浓度的戊地昔布对COX-2mRNA及蛋白的表达均没有影响,随着浓度的增加明显抑制COX-2mRNA及蛋白的表达。结论:戊地昔布可诱导人食管癌Eca109细胞发生凋亡,其诱导凋亡的机制可能与抑制COX-2的表达有关。     

Anticancer Drug-mediated Induction of Multidrug Resistance-associated Genes and Protein Kinase C Isozymes in the T-Lymphoblastoid Cell Line CCRF-CEM and in Blasts from Patients with Acute Lymphoblastic Leukemias

The major determinants mediating drug resistance in acute lymphoblastic leukemias (ALL) unresponsive to chemotherapy, are still unclear. For example, it is still unknown whether selection or induction processes are responsible for drug resistance here or whether protein kinase C (PKC) isozymes contribute to the resistant phenotype. Therefore, inducibility of resistance factors or PKC isozymes genes was examined in CCRF‐CEM cells treated with diverse anticancer drugs‐ adriamycin, camptothecin, etoposide or vincristine‐at sublethal concentrations for 24 h. MDR1, MRP1, LRP and PKC isozyme α, β₁, β₂, ε, ι, η, θ, ζ gene expression was determined by cDNA‐PCR. We found significant dose‐dependent, mostly combined, induction of the MDR1, MRP1 and LRP genes. Significantly enhanced gene expression of the majority of PKC isozyme genes was found after treatment with camptothecin. PKCζ was upregulated throughout by each anticancer drug applied in this setting. A series of selected CCRF‐CEM‐derived multidrug resistance (MDR) sublines also showed enhanced expression of the PKC isozymes compared to the parental cell line. MDR1 and PKCη gene expression levels were correlated highly significantly. Blasts from two patients with ALL during the first week of monotherapy with steroids revealed combined induction of the MDR1, multidrug resistance‐associated protein 1 (MRP1), lung cancer resistance‐related protein (LRP) and most PKC isozymes, predominantly PKCζ. Another patient with T‐ALL, who failed to respond to four months of intensive chemotherapy, showed an enhanced MRP1 gene expression combined with markedly overexpression of PKCη and PKCθ. Furthermore, the camptothecin and etoposide‐mediated induction of resistance factors in the CCRF‐CEM cell line could be suppressed by staurosporine, a rather unspecific inhibitor of protein kinases. However, selective inhibitors of PKC isozymes (bisindolylmaleimide GÖ 6850, indolocarbazole GÖ 6976) produced no significant effects here. Therefore, the PKC isozymes η, θ and ζ are of interest as potential targets to overcome drug resistance in ALL.     

Alteration of Type II Regulatory Subunit of cAMP-dependent Protein Kinase in Human Cisplatin-resistant Cells as a Basis of Collateral Sensitivity to 8-Chloro-cAMP

A cyclic adenosine 3',5'-monophosphate (cAMP) analogue, 8-chloro-cAMP (8-Cl-cAMP), had a collateral growth-inhibitory effect on a cis -diamminedichloroplatinum(II) (CDDP)-resistant human cancer cell lines (PC-14/CDDP). The non-selective analogues dibutyryl-cAMP, 8-bromo-cAMP and forskolin, which are cAMP agonists, showed far less cytotoxicity than 8-Cl-cAMP in both cell lines. There was no significant difference in cAMP content between PC-14 and PC-14/CDDP. Because 8-Cl-cAMP has been shown to bind selectively to the site I receptor of the type II regulatory subunit (RII) of cAMP-dependent protein kinase, we determined the level of expression of regulatory subunits in PC-14 and PC-14/CDDP cells by photoaffinity labeling. PC-14/CDDP cells had a higher RII level, low site I receptor of type I regulatory subunit (RI) level, and a lower RI/RII ratio than the parental PC-14 cells. Exposure to 8-Cl-cAMP increased the RI and RII level in PC-14/CDDP cells in dose- and time-dependent manners. On the other hand, in parental PC-14 cells, RII was not detected and the levels of RI and RII were not increased by exposure to 8-Cl-cAMP. These results suggested that the change in RI and/or RII levels caused by 8-Cl-cAMP was correlated with 8-Cl-cAMP-induced growth inhibition and that the collateral sensitivity to 8-Cl-cAMP in CDDP-resistant cells was due to the increased RII level. Our results suggest that 8-Cl-cAMP can be used in combination with CDDP and that measurement of RI and RII levels and/or the RI/RII ratio is a useful tool to predict CDDP sensitivity.     

抑制Src酪氨酸激酶对非小细胞肺癌细胞分泌MMP-2和MMP-9的影响

背景与目的 Src酪氨酸激酶和基质金属蛋白酶在肺癌的浸润和转移中发挥重要作用。本研究旨在探讨抑制Src酪氨酸激酶对非小细胞肺癌(non-small cell lung cancer,NSCLC)细胞分泌基质金属蛋白酶-2(matrix metalloproteinase2,MMP-2)和基质金属蛋白酶-9(matrix metalloproteinase9,MMP-9)以及NSCLC细胞侵袭浸润的影响。方法采用ELISA法检测NSCLC细胞(PC14PE6、H226、PC-9、A549)培养上清中MMP-2和MMP-9含量以及抑制Src酪氨酸激酶对NSCLC细胞分泌MMP-2和MMP-9的影响;Boyden chamber法检测抑制Src酪氨酸激酶对NSCLC细胞体外侵袭浸润的影响。结果 NSCLC细胞中PC14PE6和H226中MMP-2和MMP-9的水平较高,A549细胞中MMP-9的水平较低,而MMP-2和MMP-9在PC-9细胞中检测不到。Src酪氨酸激酶抑制剂对PC14PE6中的MMP-2水平以及PC14PE6、H226和A549细胞中的MMP-9水平呈剂量依赖性抑制关系。10μM Src酪氨酸激酶抑制剂使PC14PE6细胞中的MMP-2水平、H226细胞和A549细胞中的MMP-9水平降低50%以上。10μM Src酪氨酸激酶抑制剂对H226细胞中的MMP-2无明显抑制作用。Src酪氨酸激酶抑制剂对4种NSCLC细胞体外侵袭浸润的抑制程度略有差异,但均呈现明显的剂量依赖性抑制作用。3μM Src酪氨酸激酶抑制剂对PC14PE6、H226、A549和PC-9细胞体外侵袭浸润的抑制率分别为79.1%、68.09%、90.96%和96.98%(P<0.001)。结论通过抑制NSCLC细胞分泌MMP-2和MMP-9,抑制Src酪氨酸激酶可降低细胞的体外侵袭浸润能力。     

p16INK4 Expression Is Associated with the Increased Sensitivity of Human Non-small Cell Lung Cancer Cells to DNA Topoisomerase I Inhibitors

Inactivation of p16^INK4, an inhibitor of cyclin-dependent kinases 4 (CDK4) and 6 (CDK6), may be essential for ontogenesis in non-small cell lung cancer (NSCLC). We examined the sensitivity of two clones of P16^INK4 -transfected NSCLC cell line with homozygous deletion of p16^INK4, A549/pl6-l and 2, to DNA topoisomerase I (topo I) inhibitors. A549/pl6-l and -2 showed 7.7- and 9.1-fold increases in sensitivity to CPT-11 (11,7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin), respectively, compared with A549 cells. Ectopic p16^INK4-expressing cells also showed ∼4.0-fold increase in sensitivity to SN-38 (7-ethyl-10-hydroxycamptothecin), the active metabolite of CPT-11, compared to the parent cells. The topo I-mediated DNA relaxation activities of ectopic p16^INK4-expressing cells were approximately 5 times higher than those of the parent cells. Northern and western blot analyses indicate that these increased topo I activities of ectopic p16^INK4-expressing cells were due to an elevated topo I mRNA level and an increase in topo I protein. The chemosensitivity to topo I inhibitors, topo I mRNA level, protein content and activity of a pl6^INK4 revertant, lacking functional p16^INK4, tended to be restored toward those of the parental phenotype to some extent. These results suggest that p16^1NK4 expression is closely associated with the increased sensitivity of ectopic pl6^INK4-expressing NSCLC cells to topo I inhibitors. The up-regulation of topo I mRNA level, protein content and activity may he responsible for this hypersensitivity.