A1E inhibits proliferation and induces apoptosis in NCI-H460 lung cancer cells via extrinsic and intrinsic pathways

It has been reported that extracts from Asian traditional/medical herbs possess therapeutic agents against cancers, metabolic diseases, inflammatory diseases, and other intractable diseases. In this study, we assessed the molecular mechanisms involved in the anticancer effects of A1E, the extract of Korean medicinal herbs. We examined the role of the cytotoxic and apoptotic pathways in the cancer chemopreventive activity in non-small-cell lung cancer (NSCLC) cell lines NCI-H460 and NCI-H1299. A1E inhibited the proliferation of NCI-H460 more efficiently than NCI-H1299 (p53^?/?) cells. The apoptosis was detected by nuclear morphological changes, annexin V-FITC/PI staining, cell cycle analysis, western blot, RT-PCR, and measurement of mitochondrial membrane potential. A1E induced cellular morphological changes and nuclear condensation at 24 h in a dose-dependent manner. A1E also perturbed cell cycle progression at the sub-G1 stage and altered cell cycle regulatory factors in NCI-H460 cells. Furthermore, A1E inhibited the PI3K/Akt and NF-κB survival pathways, and it activated apoptotic intrinsic and extrinsic pathways. A1E increased the expression levels of members of the extrinsic death receptor complex FasL and FADD. In addition, A1E treatment induced cleavage of caspase-8, caspase-9, caspase-3, and poly ADP-ribose polymerase (PARP), whereas the expression levels of Bcl-2 and Bcl-xl were downregulated. A1E induced mitochondrial membrane potential collapse and cytochrome C release. Our results suggest that A1E induces apoptosis via activation of both extrinsic and intrinsic pathways and inhibition of PI3K/Akt survival signaling pathways in NCI-H460 cells. In conclusion, these data demonstrate the potential of A1E as a novel chemotherapeutic agent in NSCLC.     

Reticulocalbin 1 is required for proliferation and migration of non-small cell lung cancer cells regulated by osteoblast-conditioned medium

Reticulocalbin1 (RCN1) is implicated in tumorigenesis and tumour progression. However, whether RCN1-mediated bone metastasis of non-small cell lung cancer (NSCLC) cells was elusive. Here, we assessed the effect of osteoblast-conditioned medium (CM) on proliferation and migration of NSCLC cell line, NCI-H1299 and NCI-H460 cells, and identified the soluble mediators in CMs from osteoblasts and NSCLC cells using MTT, Clonogenicity, Transwell, wound healing, RT-PCR, and Western blotting assays, and LC-MS/MS analysis, respectively. Furthermore, the role of RCN1 was investigated in NSCLC cells cultured with or without osteoblast-CM. Tumour growth and bone resorption were measured in a nude mouse model bearing NCI-H1299 cells transduced with shRNA/RCN1 vector using in vivo imaging technique and micro-CT. The results showed that RCN1 with a higher abundance in osteoblast-CM, which was present in extracellular vesicles (EVs), enhanced RCN1 expression in NSCLC cells. Osteoblast-CM partially offset the inhibitory effect of RCN1 depletion on proliferation and migration of NSCLC cells. RCN1 depletion-induced endoplasmic reticulum (ER) stress caused by increasing GRP78, CHOP, IRE1α, p-IRE1α, p-PERK and p-JNK, which was positively regulated by self-induced autophagy, contributed to suppression of proliferation and migration in NCI-H1299 cells. Therefore, osteoblasts produced RCN1 to transfer into NSCLC cells partially through EVs, facilitating proliferation and migration of NSCLC cells via blocking ER stress. RCN1 could be required for proliferation and migration of NSCLC cells regulated by osteoblast-CM.     

Bombesin activates MAP kinase in non-small cell lung cancer cells

The effects of bombesin (BB) on mitogen activated protein (MAP) kinase were investigated using non-small cell lung cancer (NSCLC) cells. By Western blot, both 42 and 44 kDalton forms of MAP kinase were present in NCI-H1299 and NCI-H838 cells. Addition of BB to NCI-H1299 cells resulted in phosphorylation of the MAP kinase substrate myelin basic protein (MBP). Phosphorylation of MBP was maximal 6 min after the addition of 10 nM BB to NCI-H1299 cells. Addition of gastrin releasing peptide (GRP) or GRP14-27 but not GRP1-16 to NCI-H 1299 cells caused MBP phosphorylation. The effects of BB were inhibited by BW2258U89, a BB receptor antagonist, and PD98059, a MAP kinase kinase inhibitor. Also, PD98059 inhibited the clonal growth of NCI-H1299 cells. These data suggest that MAP kinase may be an important regulatory enzyme in NSCLC.     

BCI induces apoptosis via generation of reactive oxygen species and activation of intrinsic mitochondrial pathway in H1299 lung cancer cells

The compound(E)-2-benzylidene-3-(cyclohexylamino)-2,3-dihydro-1 H-inden-1-one(BCI) is known as an inhibitor of dual specific phosphatase 1/6 and mitogen-activated protein kinase. However, its precise anti-lung cancer mechanism remains unknown. In this study, the effects of BCI on the viability of non-small cell lung cancer cell lines NCI-H1299, A549, and NCI-H460 were evaluated. We confirmed that BCI significantly inhibited the viability of p53(-) NCI-H1299 cells as compared to NCI-H460 and A549 cells, which express wild-type p53. Furthermore, BCI treatment increased the level of cellular reactive oxygen species and pre-treatment of cells with N-acetylcysteine markedly attenuated BCI-mediated apoptosis of NCI-H1299 cells. BCI induced cellular morphological changes, inhibited viability, and produced reactive oxygen species in NCI-H1299 cells in a dose-dependent manner. BCI induced processing of caspase-9, caspase-3, and poly ADP-ribose polymerase as well as the release of cytochrome c from the mitochondria into the cytosol. In addition, BCI downregulated Bcl-2 expression and enhanced Bax expression in a dose-dependent manner in NCI-H1299 cells. However, BCI failed to modulate the expression of the death receptor and extrinsic factor caspase-8 and Bid, a linker between the intrinsic and extrinsic apoptotic pathways in NCI-H1299 cells. Thus, BCI induces apoptosis via generation of reactive oxygen species and activation of the intrinsic pathway in NCI-H1299 cells.     

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.     

Toward chelerythrine optimization: Analogues designed by molecular simplification exhibit selective growth inhibition in non-small-cell lung cancer cells

A series of novel chelerythrine analogues was designed and synthesized. Antitumor activity was evaluated against A549, NCI-H1299, NCI-H292, and NCI-H460 non-small-cell lung cancer (NSCLC) cell lines in vitro. The selectivity of the most active analogues and chelerythrine was also evaluated, and we compared their cytotoxicity in NSCLC cells and non-tumorigenic cell lines, including human umbilical vein endothelial cells (HUVECs) and LL24 human lung fibroblasts. In silico studies were performed to establish structure–activity relationships between chelerythrine and the analogues. The results showed that analogue compound 3f induced significant dose-dependent G₀/G₁ cell cycle arrest in A549 and NCI-H1299 cells. Theoretical studies indicated that the molecular arrangement and electron characteristics of compound 3f were closely related to the profile of chelerythrine, supporting its activity. The present study presents a new and simplified chelerythrinoid scaffold with enhanced selectivity against NSCLC tumor cells for further optimization.     

Rhamnetin and Cirsiliol Induce Radiosensitization and Inhibition of Epithelial-Mesenchymal Transition (EMT) by miR-34a-mediated Suppression of Notch-1 Expression in Non-small Cell Lung Cancer Cell Lines

Radioresistance is a major cause of decreasing the efficiency of radiotherapy for non-small cell lung cancer (NSCLC). To understand the radioresistance mechanisms in NSCLC, we focused on the radiation-induced Notch-1 signaling pathway involved in critical cell fate decisions by modulating cell proliferation. In this study, we investigated the use of Notch-1-regulating flavonoid compounds as novel therapeutic drugs to regulate radiosensitivity in NSCLC cells, NCI-H1299 and NCI-H460, with different levels of radioresistance. Rhamnetin and cirsiliol were selected as candidate Notch-1-regulating radiosensitizers based on the results of assay screening for activity and pharmacological properties. Treatment with rhamnetin or cirsiliol reduced the proliferation of NSCLC cells through the suppression of radiation-induced Notch-1 expression. Indeed, rhamnetin and cirsiliol increased the expression of tumor-suppressive microRNA, miR-34a, in a p53-dependent manner, leading to inhibition of Notch-1 expression. Consequently, reduced Notch-1 expression promoted apoptosis through significant down-regulation of the nuclear factor-κB pathway, resulting in a radiosensitizing effect on NSCLC cells. Irradiation-induced epithelial-mesenchymal transition was also notably attenuated in the presence of rhamnetin and cirsiliol. Moreover, an in vivo xenograft mouse model confirmed the radiosensitizing and epithelial-mesenchymal transition inhibition effects of rhamnetin and cirsiliol we observed in vitro. In these mice, tumor volume was significantly reduced by combinational treatment with irradiation and rhamnetin or cirsiliol compared with irradiation alone. Taken together, our findings provided evidence that rhamnetin and cirsiliol can act as promising radiosensitizers that enhance the radiotherapeutic efficacy by inhibiting radiation-induced Notch-1 signaling associated with radioresistance possibly via miR-34a-mediated pathways.     

MiR-15a-16 represses Cripto and inhibits NSCLC cell progression

MicroRNAs (miRNAs) are small noncoding RNAs that have important roles in cancer. The altered expressions of miRNAs and their target genes are frequently detected in various tumors. In this study, downregulation of miR-15a-16 in nonsmall cell lung cancer (NSCLC) was found to be inversely correlated with Cripto. Results from the Luciferase reporter assay and Western blot analysis also confirmed that Cripto is a direct target of miR-15a-16. In addition, transfection of miR-15a-16 expression plasmid inhibited the invasion ability and promoted the apoptosis of NCI-H23 and NCI-H358 cells. Moreover, miR-15a-16 overexpression suppressed tumor growth in vivo. These findings clearly suggest that the downregulation of miR-15a-16 with Cripto amplification may be involved in the development of NSCLC.     

Rapamycin decreases survivin expression to induce NSCLC cell apoptosis under hypoxia through inhibiting HIF-1α induction

Survivin is a member of the inhibitor of apoptosis protein family that is overexpressed in various tumors and is important in restricting apoptosis. Understanding the molecular events of apoptosis may provide information for developing novel therapeutic agents targeting non-small cell lung cancer (NSCLCs). This study used three human NSCLC cell lines, NCI-H1299, SK-MES-1, and NCI-H460. Changes in apoptosis, the mRNA and protein expression of survivin under normoxia and hypoxia, with or without rapamycin treatment were analyzed. In addition, siRNA and ChIP assay were further applied to demonstrate the role of hypoxia-inducible factor 1 (HIF-1)α in regulating survivin expression regulation under hypoxia during rapamycin induced NSCLC cell apoptosis. Treatment with rapamycin resulted in significantly increased NSCLC cells apoptosis under hypoxia. We demonstrated for the first time that rapamycin inhibited hypoxia-induced survivin expression in NSCLC cell lines. We further demonstrated that HIF-1α participated in hypoxia-induced survivin expression, and that rapamycin inhibited hypoxia-induced HIF-1α expression by enhancing its degradation. The results above collectively showed that rapamycin inhibits HIF-1α-induced survivin expression under hypoxia to induce NSCLC apoptosis.     

ADFP promotes cell proliferation in lung adenocarcinoma via Akt phosphorylation

Previously, we identified differentially expressed proteins, including ADFP, between lung adenocarcinoma (LAC) tissue and paired normal bronchioloalveolar epithelium. In this study, we investigated the role of ADFP in LAC. ADFP levels in the serum of patients with lung cancer and benign diseases were measured by enzyme-linked immunosorbent assays (ELISA). shRNA was used to knock-down or overexpress ADFP in A549 and NCI-H1299 cells. The biological function of ADFP and its underlying mechanisms was evaluated in vivo and in vitro. ADFP was highly expressed in the serum of lung cancer patients, especially those with LAC. ADFP promoted cell proliferation and up-regulated the p-Akt/Akt ratio in A549 and NCI-H1299 cells in vitro. Furthermore, in nude mice, ADFP promoted tumour formation with high levels of p-Akt/Akt, Ki67 and proliferating cell nuclear antigen (PCNA). Similar to the effect of ADFP knock-down, MK-2206 (a phosphorylation inhibitor of Akt) reduced A549 and NCI-H1299 cell proliferation. In ADFP-overexpressing A549 and NCI-H1299 cells, proliferation was suppressed by MK-2206 and returned to the control level. ADFP did not regulate invasion, migration or adhesion in LAC cells. Together, these results suggest that ADFP promotes LAC cell proliferation in vitro and in vivo by increasing Akt phosphorylation level.     

The antitumor effect of hinesol,extract from Atractylodes lancea (Thunb.) DC. by proliferation,inhibition, and apoptosis induction via MEK/ERK and NF-κB pathway in non–small cell lung cancer cell lines A549 and NCI-H1299

Lung cancer (especially, non–small cell lung cancer [NSCLC]) is one of the most malignant cancers in the world. Hinesol is the major component of the essential oil of Atractylodes lancea (Thunb.) DC and possesses the most promising anticancer function. However, the effects and molecular mechanism of hinesol on antiproliferation in NSCLC cells has not been well understood. In this study, we found that hinesol effectively inhibited the A549 and NCI-H1299 cells in a dose- and time-dependent manner by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide assay. In addition, hinesol induced cell cycle arrest at G0/G1 phase and apoptosis assessed by flow cytometry in A549 cells. Furthermore, Western blot analysis showed that hinesol decreased phosphorylation of mitogen-activated protein kinase, extracellular signal-regulated kinase, IκBα, and p65 inhibited the expressions of Bcl-2, cyclin D1 and upregulated the expression of Bax. Based on these results, hinesol might be a potential drug candidate of anti-NSCLC for therapy.     

Knockdown of LncRNAZFAS1 suppresses cell proliferation and metastasis in non-small cell lung cancer

ABSTRACT

To evaluate the effects of LncRNAZFAS1 on cell proliferation and tumor metastasis in non-small cell lung cancer (NSCLC), we detected the expression level of LncRNAZFAS1 in NSCLC-related tissues and cells. qRT-PCR results revealed that LncRNAZFAS1 in tumor tissues was significantly higher than that in normal lung tissue, especially significantly up-regulated in stage III / IV and in metastatic NSCLC tissues. LncRNAZFAS1 expression was dramatically up-regulated in 4 NSCLC-related cells (A549, SPC-A1, SK-MES-1, and NCI-H1299), with having the highest expression level in A549 cells. Furthermore, we implemented a knockdown of LncRNAZFAS1 in A549 cells, and the results of CCK8 and Transwell assays suggested that knockdown of LncRNAZFAS1 significantly inhibited NSCLC cell proliferation and metastasis. Next, we constructed a tumor xenograft model to evaluate the effect of LncRNAZFAS1 on the NSCLC cell proliferation in vivo. The results indicated that knockdown of LncRNAZFAS1 dramatically inhibited A549 cells proliferation and repressed tumor growth. Additionally, knockdown of LncRNAZFAS1 drastically weakened the expressions of MMP2, MMP9 and Bcl-2 proteins, whereas noticeably strengthened the expression of BAX protein. Our results altogether suggest that knockdown of LncRNAZFAS1 has a negative effect on the proliferation and metastasis of NSCLC cell, which implying LncRNAZFAS1 is a potential unfavorable biomarker in patients with NSCLC.     

Bombesin receptor subtype-3 agonists stimulate the growth of lung cancer cells and increase EGF receptor tyrosine phosphorylation

The effects of bombesin receptor subtype-3 (BRS-3) agonists were investigated on lung cancer cells. The BRS-3 agonist (DTyr⁶, (Ala¹¹, Phe¹³, Nle¹⁴) bombesin^6-14 (BA1), but not gastrin releasing peptide (GRP) or neuromedin B (NMB) increased significantly the clonal growth of NCI-H1299 cells stably transfected with BRS-3 (NCI-H1299-BRS-3). Also, BA1 addition to NCI-H727 or NCI-H1299-BRS-3 cells caused Tyr¹⁰⁶⁸ phosphorylation of the epidermal growth factor receptor (EGFR). Similarly, (DTyr⁶, R-Apa¹¹, Phe¹³, Nle¹⁴) bombesin^6-14 (BA2) and (DTyr⁶, R-Apa¹¹, 4-Cl,Phe¹³, Nle¹⁴) bombesin^6-14 (BA3) but not gastrin releasing peptide (GRP) or neuromedin B (NMB) caused EGFR transactivation in NCI-H1299-BRS-3 cells. BA1-induced EGFR or ERK tyrosine phosphorylation was not inhibited by addition of BW2258U89 (BB₂R antagonist) or PD168368 (BB₁R antagonist) but was blocked by (DNal-Cys-Tyr-DTrp-Lys-Val-Cys-Nal)NH₂ (BRS-3 ant.). The BRS-3 ant. reduced clonal growth of NCI-H1299-BRS-3 cells. BA1, BA2, BA3 and BRS-3 ant. inhibit specific ¹²⁵I-BA1 binding to NCI-H1299-BRS-3 cells with an IC₅₀ values of 1.1, 21, 15 and 750 nM, respectively. The ability of BRS-3 to regulate EGFR transactivation in NCI-H1299-BRS-3 cells was reduced by AG1478 or gefitinib (EGFR tyrosine kinase inhibitors), GM6001 (matrix metalloprotease inhibitor), PP2 (Src inhibitor), N-acetylcysteine (anti-oxidant), Tiron (superoxide scavenger) and DPI (NADPH oxidase inhibitor). These results demonstrate that BRS-3 agonists may stimulate lung cancer growth as a result of EGFR transactivation and that the transactivation is regulated by BRS-3 in a Src-, reactive oxygen and matrix metalloprotease-dependent manner.     

p53 gene status modulates the chemosensitivity of non-small cell lung cancer cells

This study examined the effects of p53 gene status on DNA damage-induced cell death and chemosensitivity to various chemotherapeutic agents in non-small cell lung cancer (NSCLC) cells. A mutant p53 gene was introduced into cells carrying the wild-type p53 gene and also vice versa to introduce the wild-type p53 gene into cells carrying the mutant p53 gene. Chemosensitivity and DNA damage-induced apoptosis in these cells were then examined. This study included five cell lines, NCI-H1437, NCI-H727, NCI-H441 and NCI-H1299 which carry a mutant p53 gene and NCI-H460 which carries a wild-type p53 gene. Mutant p53-carrying cells were transfected with the wild-type p53 gene, while mutant p53 genes were introduced into NCI-H460 cells. These p53 genes were individually mutated at amino acid residues 143, 175, 248 and 273. The representative cell line NCI-H1437 cells transfected with wild-type p53 gene (H1437/wtp53) showed a dramatic increase in susceptibility to three anticancer agents (7-fold to cisplatin, 21-fold to etoposide, and 20-fold to camptothecin) compared to untransfected or neotransfected H1437 cells. An increase in chemosensitivity was also observed in wild-type p53 transfectants of H727, H441, H1299 cells. The results of chemosensitivity were consistent with the observations on apoptotic cell death. H1437/wtp53 cells, but not H1437 parental cells, exhibited a characteristic feature of apoptotic cell death that generated oligonucleosomal-sized DNA fragments. In contrast, loss of chemosensitivity and lack of p53-mediated DNA degradation in response to anticancer agents were observed in H460 cells transfected with mutant p53. These observations suggest that the increase in chemosensitivity was attributable to wild-type p53 mediation of the process of apoptosis. In addition, our results also suggest that p53 gene status modulates the extent of chemosensitivity and the induction of apoptosis by different anticancer agents in NSCLC cells.     

ICAM-3 endows anticancer drug resistance against microtubule-damaging agents via activation of the ICAM-3-AKT/ERK-CREB-2 pathway and blockage of apoptosis

In a previous study, we showed that induction of ICAM-3 endows radioresistance in cervical cancer [1]. To ascertain whether ICAM-3 also promotes anticancer drug resistance, mock control (H1299/pcDNA3) or ICAM-3-expressing stable transfectants (H1299/ICAM-3) of the non-small cell lung cancer (NSCLC) cell line, NCI-H1299, were generated and treated with the microtubule-damaging agents, paclitaxel (TXL) and vincristine (VCS). TXL-/VCS-treated H1299/ICAM-3 cells showed significantly lower levels of apoptosis, activation of caspases-3, 8 or 9, and decrease in anti-apoptotic protein levels, compared to H1299/pcDNA3 cells. Our data clearly indicate that ICAM-3 promotes drug resistance via inhibition of apoptosis. We additionally showed that Akt, ERK, and CREB-2 are located downstream of ICAM-3, and activation of the ICAM-3-Akt/ERK-CREB-2 pathway induces resistance against TXL and VCS. ICAM-3-expressing stable NCI-H460/ICAM-3 transfectant cells and radioresistant SiHa cells endogenously overexpressing ICAM-3 additionally showed drug resistance against TXL and VCS via activation of the ICAM-3-Akt/ERK-CREB-2 pathway. The finding that ICAM-3 endows drug resistance as well as radioresistance supports its potential utility as a major therapeutic target against cancer.     

Taxol-induced reorganization of the microtubule system in murine splenic lymphocytes inhibits response to allogeneic cells but not to concanavalin A

The exposure of mouse splenic lymphocytes to the microtubule assembly-promoting drug taxol (10 microM for 4 h) results in an extensive reorganization of the microtubule system to form one to a few large bundles of microtubules, which extend from the centrosome. Lymphocytes pretreated with taxol for 4 h, or cultured in the continued presence of taxol, respond normally to the mitogen concanavalin A up to, and including, the stage of DNA replication. In contrast, the induction of DNA synthesis during the alloactivation of lymphocytes is inhibited when taxol is present in the mixed leukocyte culture. If the stimulators are pretreated with this drug, the mixed leukocyte reaction occurs normally, but pretreatment of the responders inhibits the proliferative response markedly. Microscopic observations of nuclear morphologies in these populations and autoradiography indicate that taxol inhibition occurs early in alloactivation, prior to DNA replication. The responding ability of taxol-treated lymphocytes is not restored to control levels by the addition of interleukin 2, leading to the suggestion that interleukin 2 receptors do not emerge or function normally in these cells. We conclude that the capacity to respond to allogeneic cells, but not to a mitogen, is dependent on the presence of the normal submembranous organization of the microtubule system.     

Inhibition of human non-small cell lung cancers with a targeted cytotoxic somatostatin analog, AN-162

Human non-small cell lung cancers (NSCLCs) express receptors for somatostatin. The cytotoxic analog of somatostatin AN-162 (AEZS-124), consisting of doxorubicin linked to a somatostatin analog RC-121 binds to receptors for somatostatin and is targeted to tumors expressing these receptors. The aim of this study was to investigate the effect of targeted cytotoxic somatostatin analog AN-162 on a panel of human NSCLC cell lines (A549, H460, H838, H1299) in vitro (at 0.5–100 μM concentrations) and in vivo on H460 and H1299 NSCLCs xenografted into nude mice (at the dose of 2.5 μmol/kg, i.v., once a week). The expression of mRNA for somatostatin receptor subtypes was investigated by RT-PCR in cell lines and tumor tissues. Somatostatin receptor proteins were also characterized by ligand competition assay and Western blotting. AN-162 significantly decreased cell proliferation in vitro and tumor growth (p < 0.05 vs. all groups) of H460 and H1299 NSCLCs in vivo. Based on real-time PCR array data, AN-162 induced several apoptosis-related genes in vivo in both models. Our results suggest that cytotoxic somatostatin analog AN-162 (AEZS-124) should be considered for the further development of a therapy of patients with NSCLC.     

PACAP-27 tyrosine phosphorylates mitogen activated protein kinase and increases VEGF mRNAs in human lung cancer cells

The effects of pituitary adenylate cyclase activating polypeptide (PACAP) on human lung cancer cell line NCI-1299 mitogen activated protein kinase (MAPK) tyrosine phosphorylation and vascular endothelial cell growth factor (VEGF) expression were investigated. PACAP-27 (100 nM) increased MAPK tyrosine phosphorylation 3-fold, 5 min after addition to NCI-H1299 cells. PACAP caused tyrosine phosphorylation in a concentration-dependent manner being half-maximal at 10 nM PACAP-27. PACAP-27 or PACAP-38 (100 nM) but not PACAP28-38 or VIP caused increased MAPK tyrosine phosphorylation using NCI-H1299 cells. Also, the increase in MAPK tyrosine phosphorylation caused by PACAP-27 was totally inhibited by 10 microM PACAP(6-38), a PAC(1) receptor antagonist or 10 microM PD98059, a MAPKK inhibitor. These results suggest that PAC(1) receptors regulate tyrosine phosphorylation of MAPK in a MAPKK-dependent manner. PACAP-27 (100 nM) caused increased VEGF mRNA in NCI-H1299 cells after 8 h. The increase in VEGF mRNA caused by PACAP-27 was partially inhibited by PACAP(6-38), PD98059 and H-89. Addition of VIP to NCI-H1299 cells caused increased VEGF mRNA, which was totally inhibited by H89, a PKA inhibitor. These results suggest that PAC(1) and VPAC(1) receptors regulate VEGF expression in lung cancer cells.     

Prediction of a non-small cell lung cancer sensitivity to cisplatin and paclitaxel based on the marker genes expression

The goal of the present study was to define gene expression signatures that predict a chemosensitivity of non-small cell lung cancer (NSCLC) to cisplatin and paclitaxel. To generate set of candidate genes likely to be predictive a current knowledge of the pathways involved in resistance and sensitivity to individual drugs was used. Forty four genes coding proteins belonging to following categories: ATP-dependent transport proteins, detoxification system proteins, reparation system proteins, tubulin and proteins responsible for its synthesis, cell cycle and apoptosis proteins were considered. Eight NSCLC cell lines (A549, Calul, H1299, H322, H358, H460, H292, and H23) were used in our study. For each NSCLC cell line a cisplatin and paclitaxel chemosensitivity as well as an expression level of 44 candidate genes were evaluated. To develop a chemosensitivity prediction model based on selected genes expression level a multiple regression analysis was performed. The model based on the expression level of 11 genes (TUBB3, TXR1, MRP5, MSH2, ERCC1, STMN, SMAC, FOLR1, PTPN14, HSPA2, GSTP1) allowed us to predict the paclitaxel cytotoxic concentration with high level of correlation (r = 0.91, p < 0.01). However, none model developed was able to reliably predict a sensitivity of the NSCLC cells to cisplatin.