Adenine nucleotides inhibit proliferation of the human lung adenocarcinoma cell line LXF-289 by activation of nuclear factor kappaB1 and mitogen-activated protein kinase pathways

Extracellular nucleotides have a profound role in the regulation of the proliferation of diseased tissue. We studied how extracellular nucleotides regulate the proliferation of LXF-289 cells, the adenocarcinoma-derived cell line from human lung bronchial tumor. ATP and ADP strongly inhibited LXF-289 cell proliferation. The nucleotide potency profile was ATP = ADP = ATPgammaS > > UTP, UDP, whereas alpha,beta-methylene-ATP, beta,gamma-methylene-ATP, 2',3'-O-(4-benzoylbenzoyl)-ATP, AMP and UMP were inactive. The nucleotide potency profile and the total blockade of the ATP-mediated inhibitory effect by the phospholipase C inhibitor U-73122 clearly show that P2Y receptors, but not P2X receptors, control LXF-289 cell proliferation. Treatment of proliferating LXF-289 cells with 100 microm ATP or ADP induced significant reduction of cell number and massive accumulation of cells in the S phase. Arrest in S phase is also indicated by the enhancement of the antiproliferative effect of ATP by coapplication of the cytostatic drugs cisplatin, paclitaxel and etoposide. Inhibition of LXF-289 cell proliferation by ATP was completely reversed by inhibitors of extracellular signal related kinase-activating kinase/extracellular signal related kinase 1/2 (PD98059, U0126), p38 mitogen-activated protein kinase (SB203508), phosphatidylinositol-3-kinase (wortmannin), and nuclear factor kappaB1 (SN50). Western blot analysis revealed transient activation of p38 mitogen-activated protein kinase, extracellular signal-related kinase 1/2, and nuclear factor kappaB1 and possibly new formation of p50 from its precursor p105. ATP-induced attenuation of LXF-289 cell proliferation was accompanied by transient translocation of p50 nuclear factor kappaB1 and extracellular signal-related kinase 1/2 to the nucleus in a similar time period. In summary, inhibition of LXF-289 cell proliferation is mediated via P2Y receptors by activation of multiple mitogen-activated protein kinase pathways and nuclear factor kappaB1, arresting the cells in the S phase.     

Lipoxygenase inhibitors abolish proliferation of human pancreatic cancer cells.

Epidemiologic and animal studies have linked pancreatic cancer growth with fat intake, especially unsaturated fats. Arachidonic acid release from membrane phospholipids is essential for tumor cell proliferation. Lipoxygenases (LOX) constitute one pathway for arachidonate metabolism, but their role in pancreatic cancer growth is unknown. The expression of 5-LOX and 12-LOX as well as their effects on cell proliferation was investigated in four human pancreatic cancer cell lines (PANC-1, MiaPaca2, Capan2, and ASPC-1). Expression of 5-LOX and 12-LOX mRNA was measured by nested RT-PCR. Effects of LOX inhibitors and specific LOX antisense oligonucleotides on pancreatic cancer cell proliferation were measured by (3)H-thymidine incorporation. Our results showed that (1) 5-LOX and 12-LOX were expressed in all pancreatic cancer cell lines tested, while they were not detectable in normal human pancreatic ductal cells; (2) both LOX inhibitors and LOX antisense markedly inhibited cell proliferation in a concentration-dependent and time-dependent manner; (3) the 5-LOX and 12-LOX metabolites 5-HETE and 12-HETE as well as arachidonic and linoleic acids directly stimulated pancreatic cancer cell proliferation; (4) LOX inhibitor-induced growth inhibition was reversed by 5-HETE and 12-HETE. The current studies indicate that both 5-LOX and 12-LOX expression is upregulated in human pancreatic cancer cells and LOX plays a critical role in pancreatic cancer cell proliferation. LOX inhibitors may be valuable for the treatment of pancreatic cancer.     

The effect of tyrphostins AG494 and AG1478 on the autocrine growth regulation of A549 and DU145 cells

We employed two selective EGFR tyrosine kinase inhibitors: AG494 (reversible) and AG1478 (irreversible) for growth regulation of human lung (A549) and prostate (DU145) cancer cell lines, cultured in chemically defined DMEM/F12 medium. Both tested tyrphostins significantly inhibited autocrine growth of the investigated cell lines. The action of AG494 was dose dependent, and at highest concentrations led to complete inhibition of growth. AG1478 seemed to be more effective at lower concentrations, but was unable to completely inhibit growth of A549 cells. Inhibition of EGFR kinase activity by AG494 in contrast to AG1478 had no effect on the activity of ERK in both cell lines. Both EGFR's inhibitors induced apoptosis of the investigated lung and prostate cancer cell lines, but the proapoptotic effect of the investigated tyrphostins was greater in A549 than in DU145 cells. The tyrphostins arrested cell growth of DU145 and A549 cells in the G1 phase, similarly to other known inhibitors of EGFR. The influence of AG494 and AG1478 on the activity of two signaling proteins (AKT and ERK) was dependent upon the kind of investigated cells. In the case of DU145 cells, there was an evident decline in enzymatic activity of both kinases (stronger for AG1478), while in A549, only AG1478 effectively inhibited the phosphorylation of Akt. Tyrphostins AG494 and AG1478 are ATP-competitors and are supposed to have a similar mechanism of action, but our results suggest that this is not quite true.     

Anti-proliferative effect of cytohesin inhibition in gefitinib-resistant lung cancer cells

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKI), such as gefitinib, have been proven to efficiently inhibit the proliferation of a subset of non small-cell lung cancers (NSCLC). Unfortunately, the majority of NSCLC expressing wild type EGFR is primarily resistant to EGFR-TKI treatment. Here, we show that the proliferation of the gefitinib-resistant NSCLC cell lines H460 and A549 is reduced by the small molecule SecinH3 which indirectly attenuates EGFR activation by inhibition of cytohesins, a class of recently discovered cytoplasmic EGFR activators. SecinH3 and gefitinib showed a synergistic antiproliferative effect, which correlated with a profound inhibition of Akt activation and survivin expression. Treating mice bearing H460 xenografts with SecinH3 showed the antiproliferative and pro-apoptotic effect of SecinH3 in vivo. Our data suggest that targeting the EGFR indirectly by inhibiting its cytoplasmic activators, the cytohesins, has the potential to improve the treatment of primarily EGFR-TKI resistant lung cancers.     

GHRH antagonist MZ-5-156 increases the expression of AMPK in A549 lung cancer cells

AMP-activated protein kinase (AMPK) regulates cellular proliferation, growth and metabolism. Targeted activation of AMPK is considered an important therapeutic strategy for cancer treatment. To evaluate the effect of growth hormone-releasing hormone (GHRH) and its antagonist MZ-5-156 on the phosphorylation of AMPK and other related regulatory intracellular proteins we employed human non-small cell lung cancer cell line A549, which expresses GHRH receptors. Treatment of A549 cells with GHRH antagonist decreased cell proliferation and activated AMPK as well as glycogen synthase kinase (GSK)3β. Furthermore, MZ-5-156 inhibited Akt, the mammalian target of rapamycin (mTOR) and its downstream target eIF4E which controls protein synthesis and cell growth. GHRH(1-29)NH2 counteracted all these effects. HeLa human endometrial cancer cells which do not express any GHRH receptors were used as a negative control and GHRH did not induce the AMPK activation in these cells. Our results demonstrate for the first time that GHRH antagonists can regulate the AMPK metabolic pathway, which is crucial for the growth of non-small cell lung cancer and other major cancers.     

Differential effects of mesenchymal stem cells on a heterogeneous cell population within lung cancer cell lines

Although mesenchymal stem cells (MSCs) promote lung cancer growth in vivo, in vitro studies indicate that they inhibit the proliferation of lung cancer cells. Because malignant tumors contain a heterogeneous cell population with variable capacity for self-renewal, the aim of this study was to determine whether the inconsistencies between in vitro and in vivo studies are a result of differential effects of MSCs on the heterogeneous cell population within lung cancer cell lines. Human MSCs were isolated from the bone marrow, and their cell surface antigen expression and multi-lineage differentiation capacity was examined at passage 10. CD133+ cells were isolated from A549 and H446 cell lines using immunomagnetic separation. The effects of MSCs on the growth and microsphere formation of heterogeneous cell populations within two lung cancer cell lines (A549 and H446) were compared. MSCs inhibited the in vitro proliferation of both cell lines, but significantly accelerated tumor formation and stimulated tumor growth in vivo (P < 0.05). In CD133+ cells isolated from both A549 and H446 cells, co-culture with MSCs for 1–3 days significantly increased their proliferation (P < 0.05). MSCs also significantly increased microsphere formation in both cell lines (P < 0.05). Selective stimulation of CD133+ cell growth may account for the discrepant effects of MSCs on lung cancer progression.     

The transmembrane adaptor Cbp/PAG1 controls the malignant potential of human non-small cell lung cancers that have c-src upregulation

The tyrosine kinase c-Src is upregulated in various human cancers, although the precise regulatory mechanism underlying this upregulation is unclear. We previously reported that a transmembrane adaptor Csk-binding protein (Cbp; PAG1) plays an important role in controlling the cell transformation that is induced by the activation of c-Src. To elucidate the in vivo role of Cbp, we examined the function of Cbp in lung cancer cell lines and tissues. In this study, we found that Cbp was markedly downregulated in human non-small cell lung cancer (NSCLC) cells. The ectopic expression of Cbp suppressed the anchorage-independent growth of the NSCLC cell lines (A549 and Lu99) that had upregulated c-Src, whereas the Cbp expression had little effect on other NSCLC cell lines (PC9 and Lu65) that express normal levels of c-Src. The expression of Cbp suppressed the kinase activity of c-Src in A549 cells by recruiting c-Src and its negative regulator, C-terminal Src kinase (Csk), to lipid rafts. The treatment with Src inhibitors, such as PP2, dasatinib, and saracatinib, also suppressed the growth of A549 cells. Furthermore, Cbp expression attenuated the ability of A549 cells to form tumors in nude mice, invade in vitro, and metastasize in vivo. In addition, we found a significant inverse correlation between the level of Cbp expression and the extent of lymph node metastasis in human lung cancers. These results indicate that Cbp is required for the Csk-mediated inactivation of c-Src and may control the promotion of malignancy in NSCLC tumors that are characterized by c-Src upregulation.     

Pleckstrin Homology Domain of Akt Kinase: A Proof of Principle for Highly Specific and Effective Non-Enzymatic Anti-Cancer Target

While pharmacological inhibition of Akt kinase has been regarded as a promising anti-cancer strategy, most of the Akt inhibitors that have been developed are enzymatic inhibitors that target the kinase active site of Akt. Another key cellular regulatory event for Akt activation is the translocation of Akt kinase to the cell membrane from the cytoplasm, which is accomplished through the pleckstrin homology (PH) domain of Akt. However, compounds specifically interacting with the PH domain of Akt to inhibit Akt activation are currently limited. Here we identified a compound, lancemaside A (LAN-A), which specifically binds to the PH domain of Akt kinase. First, our mass spectra analysis of cellular Akt kinase isolated from cells treated with LAN-A revealed that LAN-A specifically binds to the PH domain of cellular Akt kinase. Second, we observed that LAN-A inhibits the translocation of Akt kinase to the membrane and thus Akt activation, as examined by the phosphorylation of various downstream targets of Akt such as GSK3β, mTOR and BAD. Third, in a co-cultured cell model containing human lung epithelial cancer cells (A549) and normal human primary lung fibroblasts, LAN-A specifically restricts the growth of the A549 cells. LAN-A also displayed anti-proliferative effects on various human cancer cell lines. Finally, in the A549-luciferase mouse transplant model, LAN-A effectively inhibited A549 cell growth with little evident cytotoxicity. Indeed, the therapeutic index of LAN-A in this mouse model was >250, supporting that LAN-A is a potential lead compound for PH domain targeting as a safe anti-cancer Akt inhibitor.     

Glycogen Synthase Kinase 3 Protein Kinase Activity Is Frequently Elevated in Human Non-Small Cell Lung Carcinoma and Supports Tumour Cell Proliferation

Background

Glycogen synthase kinase 3 (GSK3) is a central regulator of cellular metabolism, development and growth. GSK3 activity was thought to oppose tumourigenesis, yet recent studies indicate that it may support tumour growth in some cancer types including in non-small cell lung carcinoma (NSCLC). We examined the undefined role of GSK3 protein kinase activity in tissue from human NSCLC.

Methods

The expression and protein kinase activity of GSK3 was determined in 29 fresh frozen samples of human NSCLC and patient-matched normal lung tissue by quantitative immunoassay and western blotting for the phosphorylation of three distinct GSK3 substrates in situ (glycogen synthase, RelA and CRMP-2). The proliferation and sensitivity to the small-molecule GSK3 inhibitor; CHIR99021, of NSCLC cell lines (Hcc193, H1975, PC9 and A549) and non-neoplastic type II pneumocytes was further assessed in adherent culture.

Results

Expression and protein kinase activity of GSK3 was elevated in 41% of human NSCLC samples when compared to patient-matched control tissue. Phosphorylation of GSK3α/β at the inhibitory S21/9 residue was a poor biomarker for activity in tumour samples. The GSK3 inhibitor, CHIR99021 dose-dependently reduced the proliferation of three NSCLC cell lines yet was ineffective against type II pneumocytes.

Conclusion

NSCLC tumours with elevated GSK3 protein kinase activity may have evolved dependence on the kinase for sustained growth. Our results provide further important rationale for exploring the use of GSK3 inhibitors in treating NSCLC.     

SR48692 inhibits non-small cell lung cancer proliferation in an EGF receptor-dependent manner

Aims

The mechanism by which SR48692 inhibits non-small cell lung cancer (NSCLC) proliferation was investigated.

Main methods

The ability of SR48692 to inhibit the proliferation of NSCLC cell lines NCI-H1299 and A549 was investigated in vitro in the presence or absence of neurotensin (NTS). The ability of NTS to cause epidermal growth factor receptor (EGFR) transactivation was investigated by Western blot using NSCLC cells and various inhibitors. The growth effects and Western blot results were determined in cell lines treated with siRNA for NTSR1.

Key findings

Treatment of A549 or NCI-H1299 cells with siRNA for NTSR1 reduced significantly NTSR1 protein and the ability of SR48692 to inhibit the proliferation of A549 or NCI-H1299 NSCLC cells. Treatment of A549 and NCI-H1299 cells with siRNA for NTSR1 reduced the ability of NTS to cause epidermal growth factor receptor (EGFR) transactivation. SR48692 or gefitinib (EGFR tyrosine kinase inhibitor) inhibited the ability of NTS to cause EGFR and ERK tyrosine phosphorylation. NTS transactivation of the EGFR was inhibited by GM6001 (matrix metalloprotease inhibitor), Tiron (superoxide scavenger) or U73122 (phospholipase C inhibitor) but not H89 (PKA inhibitor). NTS stimulates whereas SR48692 or gefitinib inhibits the clonal growth of NSCLC cells.

Significance

These results suggest that SR48692 may inhibit NSCLC proliferation in an EGFR-dependent mechanism.     

MicroRNA-567 inhibits cell proliferation and induces cell apoptosis in A549 NSCLC cells by regulating cyclin-dependent kinase 8

MicroRNA-567 (miR-567) plays a decisive role in cancers whereas its role in non-small cell lung cancer (NSCLC) is still unexplored. This study was therefore planned to explore the regulatory function of miR-567 in A549 NSCLC cells and investigate its possible molecular mechanism that may help in NSCLC treatment. In the current study, miR-567 expression was examined by quantitative real time-polymerase chain reaction (qRT-PCR) in different NSCLC cell lines in addition to normal cell line. A549 NSCLC cells were transfected by miR-567 mimic, miR-567 inhibitor, and negative control siRNA. Cell proliferation was evaluated by MTT and 5-bromo-2′deoxyuridine assays. Cell cycle distribution and apoptosis were studied by flow cytometry. Bioinformatics analysis programs were used to expect the putative target of miR-567. The expression of cyclin-dependent kinase 8 (CDK8) gene at mRNA and protein levels were evaluated by using qRT-PCR and western blotting. Our results found that miR-567 expressions decreased in all the studied NSCLC cells as compared to the normal cell line. A549 cell proliferation was suppressed by miR-567 upregulation while cell apoptosis was promoted. Also, miR-567 upregulation induced cell cycle arrest at sub-G1 and S phases. CDK8 was expected as a target gene of miR-567. MiR-567 upregulation decreased CDK8 mRNA and protein expression while the downregulation of miR-567 increased CDK8 gene expression. These findings revealed that miR-567 may be a tumor suppressor in A549 NSCLC cells through regulating CDK8 gene expression and may serve as a novel therapeutic target for NSCLC treatment.     

Modulation of sodium transport in fetal alveolar epithelial cells by oxygen and corticosterone

The involvement of P2Y receptors, which are activated by extracellular nucleotides, in proliferative regulation of human lung epithelial cells is unclear. Here we show that extracellular ATP and UTP stimulate bromodeoxyuridine (BrdU) incorporation into epithelial cell lines. The nucleotide efficacy profile [ATP = ADP > UDP >or= UTP > adenosine >or= 2-methylthioadenosine-5'-diphosphate, with alpha,beta-methylene adenosine 5'-triphosphate, 2',3'-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate, AMP, UMP, and ATPalphaS inactive] and PCR analysis indicate involvement of P2Y2 and P2Y6 receptors. The signal transduction pathway, which, via the P2Y2 receptor, transmits the proliferative activity of ATP or UTP in A549 cells downstream of phospholipase C, depends on Ca2+/calmodulin-dependent protein kinase II and nuclear factor-kappaB, but not on protein kinase C. Signaling does not involve the mitogen-activated protein kinases extracellular signal-regulated kinases-1 and -2, the phosphatidylinositol 3-kinase pathway, or Src kinases. Thus nucleotides regulate proliferation of human lung epithelial cells by a novel pathway. The stimulatory effect of UTP, but not ATP, in A549 cells is attenuated by preincubation with interleukin-1beta and interleukin-6, but not tumor necrosis factor-alpha. This indicates an important role for the pyrimidine-activated P2Y receptor in the inflammatory response of lung epithelia. ATP antagonizes the antiproliferative effect of the anticancer drugs paclitaxel and etoposide, whereas it enhances the activity of cisplatin about fourfold. Thus pathways activated by extracellular nucleotides differentially control proliferation of lung epithelial tumor cells.     

Down-regulation of human complement factor H sensitizes non-small cell lung cancer cells to complement attack and reduces in vivo tumor growth

Malignant cells are often resistant to complement activation through the enhanced expression of complement inhibitors. In this work, we examined the protective role of factor H, CD46, CD55, and CD59 in two non-small cell lung cancer cell lines, H1264 and A549, upon activation of the classical pathway of complement. Complement was activated with polyclonal Abs raised against each cell line. After blocking factor H activity with a neutralizing Ab, C3 deposition and C5a release were more efficient. Besides, a combined inhibition of factor H and CD59 significantly increased complement-mediated lysis. CD46 and CD55 did not show any effect in the control of complement activation. Factor H expression was knockdown on A549 cells using small interfering RNA. In vivo growth of factor H-deficient cells in athymic mice was significantly reduced. C3 immunocytochemistry on explanted xenografts showed an enhanced activation of complement in these cells. Besides, when mice were depleted of complement with cobra venom factor, growth was recovered, providing further evidence that complement was important in the reduction of in vivo growth. In conclusion, we show that expression of the complement inhibitor factor H by lung cancer cells can prevent complement activation and improve tumor development in vivo. This may have important consequences in the efficiency of complement-mediated immunotherapies.     

Akt2 and nucleophosmin/B23 function as an oncogenic unit in human lung cancer cells

The signaling network of protein kinase B(PKB)/Akt has been implicated in survival of lung cancer cells. However, understanding the relative contribution of the different isoform of Akt network is nontrival. Here, we report that Akt2 is highly expressed in human lung adenocarcinoma cell line A549 cells. Suppression of Akt2 expression in A549 cells results in notable inhibition of cell poliferation, soft agar growth, and invasion, accompanying by a decrease of nucleophosmin/B23 protein. Overexpression of Akt1 restores cancerous growth of A549 cells in B23-knockdown (KD) cells while Akt2 overexpression did not restore proliferating potential in cells with downregulated B23, thus suggesting Akt2 requires B23 to drive proliferation of lung cancer cell. Loss of functional Akt2 and B23 has similar defects on cell proliferation, apoptotic resistance and cell cycle regulation, while loss of Akt1 has less defects on cell proliferation, survial and cell cycle progression in A549 cells. Moreover, overexpression of B23 rescues the proliferative block induced as a consequence of loss of Akt2. Thus our data suggest that Akt2/B23 functions as an oncogenic unit to drive tumorigenesis of A549 lung cancer cells.     

融合细胞株Eahy926与亲本人肺腺癌细胞株A549差异表达蛋白的蛋白质组学分析

融合细胞株Eahy926是人肺腺癌细胞株A549和人脐静脉内皮细胞杂交而成的永生化细胞株,具有血管内皮细胞的特性,已广泛用于内皮细胞相关研究. 本研究应用蛋白质组学技术分析融合细胞株Eahy926与亲本人肺腺癌细胞株A549的蛋白质差异表达,探讨融合细胞生物学特性变化及其机制. 对Eahy926和亲本A549的细胞总蛋白质进行双向凝胶电泳,在PDQuest软件辅助下找出差异表达蛋白质点,经肽质量指纹图谱(peptide mass fingerprinting, PMF) 和串级质谱(tandem mass spectrometry,TMS) 分析,SWISS-PROT数据库检索,成功鉴定出28个差异蛋白,如CATB、CK8、CK18、annexin A2、GRP78、HSP90、HSP60、vimentin等一些与分子伴侣、氧化应激、能量代谢、信号转导等有关,并与肿瘤细胞分裂增殖、分化凋亡、侵袭转移、免疫逃逸以及肿瘤血管生长密切关联的蛋白质. 研究发现,融合细胞株Eahy926和人肺腺癌细胞株A549的蛋白质组表达谱存在明显差异,这将有助于今后进一步探讨肿瘤细胞与内皮细胞的相互作用机制及其融合细胞的特性,筛选肿瘤增殖和转移相关蛋白质及分子标志物,亦可为肿瘤的抗血管生成治疗提供新思路.     

肿瘤坏死因子α和β对电离辐射诱导细胞凋亡的效应

为探讨肿瘤坏死因子（tumor necrosis foctor)α和β（TNFα和β）对电离辐射诱发细胞凋亡的效应及其机理,采用DNA琼脂糖凝胶电泳和FACS分析等方法,观察了人肿瘤坏死因子α（hTNFα)和β（hTNFβ)对^60Co-γ射线诱发细胞凋亡的形态学,生化学变化。结果显示：hTNFα或hTNFβ均可明显抑制^60Co-γ射线诱发正常人胚肺二倍体细胞（2BS）的凋亡,而相同剂量的hTNFα能促进^60Co-γ射线诱发的人体肺腺癌细胞系A549细胞凋亡,而对另一株人体肺癌SPC细胞的效应比A549降低1倍;hTNFβ能分别增强A549和SPC的细胞凋亡频率。由此认为,hTNFα和hTNFβ均可通过调节细胞的生理生化反应来改变细胞对电离辐射的敏感性,可保护正常细胞免受辐射损伤,而增加某些肿瘤细胞对辐射的敏感性。     

牛至油体外抗肿瘤活性研究

目的观察牛至油对肿瘤细胞株的生长抑制作用。方法采用MTT法检测不同浓度的牛至油对体外培养的多种肿瘤细胞株的生长抑制作用,计算半数抑制浓度(IC50)。结果不同浓度牛至油作用后,人肝癌细胞系HepG2、人子宫颈癌细胞系JTC-26和肺癌细胞系A549出现增殖阻滞。MTT法确定牛至油对肝癌HepG2的IC50为118μg/ml;人子宫颈癌JTC-26的IC50为118μg/ml;肺癌A549的IC50为59μg/ml。结论牛至油具有体外抗肿瘤活性。     

Targeting Non-Small Cell Lung Cancer Cells by Dual Inhibition of the Insulin Receptor and the Insulin-Like Growth Factor-1 Receptor

Phase III trials of the anti-insulin-like growth factor-1 receptor (IGF1R) antibody figitumumab in non-small cell lung cancer (NSCLC) patients have been discontinued owing to lack of survival benefit. We investigated whether inhibition of the highly homologous insulin receptor (IR) in addition to the IGF1R would be more effective than inhibition of the IGF1R alone at preventing the proliferation of NSCLC cells. Signalling through IGF1R and IR in the NSCLC cell lines A549 and Hcc193 was stimulated by a combination of IGF1, IGF2 and insulin. It was inhibited by antibodies that block ligand binding, αIR3 (IGF1R) and IR47-9 (IR), and by the ATP-competitive small molecule tyrosine kinase inhibitors AZ12253801 and NVPAWD742 which inhibit both IGF1R and IR tyrosine kinases. The effect of inhibitors was determined by an anchorage-independent proliferation assay and by analysis of Akt phosphorylation. In Hcc193 cells the reduction in cell proliferation and Akt phosphorylation due to anti-IGF1R antibody was enhanced by antibody-mediated inhibition of the IR whereas in A549 cells, with a relatively low IR:IGF1R expression ratio, it was not. In each cell line proliferation and Akt phosphorylation were more effectively inhibited by AZ12253801 and NVPAWD742 than by combined αIR3 and IR47-9. When the IGF1R alone is inhibited, unencumbered signalling through the IR can contribute to continued NSCLC cell proliferation. We conclude that small molecule inhibitors targeting both the IR and IGF1R more effectively reduce NSCLC cell proliferation in a manner independent of the IR:IGF1R expression ratio, providing a therapeutic rationale for the treatment of this disease.     

Synthesis and inhibition of cancer cell proliferation of (1,3')-bis-tetrahydroisoquinolines and piperazine systems

Some (1,3')-bis-tetrahydroisoquinolines were reported as scaffold intermediates for the synthesis of pentacyclic piperazine core alkaloids and their cytotoxicity against cancerous cell lines was evaluated. The NMR and X-ray structural assignments revealed an anti C3-C11 backbone stereochemistry of piperazine structures. Inhibition of cancer cell proliferation of (1,3')-bis-tetrahydroisoquinoline scaffolds and pentacyclic piperazine systems was assessed against three human cancer cell lines (K562 myelogenous leukemia, A549 lung carcinoma, MCF-7 breast adenocarcinoma) and both mouse tumor cell lines of blood (P388) and lymphocytic (L1210) leukemia with considerable activity against the latter. The cell cycle analysis was also studied by flow cytometry measurement on K562 cell line.     

Decreased expression of the type I isozyme of cAMP-dependent protein kinase in tumor cell lines of lung epithelial origin

A spontaneous transformant derived from a mouse lung epithelial cell line exhibited decreased cAMP-dependent protein kinase (PKA) activity. DEAE column chromatography demonstrated that this was caused by specific loss of the type I PKA isozyme (PKA I). Western immunoblot analysis indicated that indeed several mouse lung tumor-derived cell lines and spontaneous transformants of immortalized, nontumorigenic lung cell lines contained less PKA I regulatory subunit (RI) protein than normal cell lines. PKA II regulatory subunit protein differed only slightly among cell lines and showed no conspicuous trend between normal and neoplastic cells. The decrease in RI was apparently concomitant with decreased catalytic (C) subunit levels in neoplastic cells since no free catalytic subunit activity was detected by DEAE chromatography. Northern blot analysis using RI alpha and C alpha cDNA probes showed that the levels of RI alpha and C alpha mRNAs paralleled their intracellular protein concentrations; neoplastic cell lines contained significantly less RI alpha and C alpha mRNAs than the normal cell line. The decreased expression of both RI and C subunits therefore results in a net decrease of PKA I in neoplastic lung cells, an isozymic difference which may account for the differential effects of cAMP analogs on cell growth and differentiation in normal and neoplastic cells.