Lung Tumor Cell Growth Research Articles

Abstract 4372: Phosphodiesterase 10A inhibition suppresses lung tumor cell growth by activating PKG to inhibit ras and Wnt signaling

Abstract We recently reported that phosphodiesterase 10A (PDE10) is overexpressed in colon tumors and essential for colon tumor cell growth (Li et al., Oncogene 2014), but a role in lung cancer has not been well studied. Human non-small cell lung cancer (NSCLC) cell lines were found to express appreciably higher PDE10 levels compared with normal airway epithelial cells, while silencing of PDE10 expression with siRNA or inhibition of PDE10 activity with small molecule inhibitors selectively inhibits NSCLC cell growth. Here we study the mechanism by which PDE10 inhibitors suppress NSCLC cell growth. At concentrations effective for inhibiting NSCLC cell growth, the PDE10 inhibitor, PQ10 rapidly increases intracellular cGMP levels as determined by a luminescence biosensor assay. Within the same concentration range and time period, PQ10 activates PKA and PKG, demonstrated by an increase in phosphorylation of the substrate, VASP at Ser157 and Ser239, respectively. Under the same conditions, PQ10 suppresses the phosphorylation of Erk1/2 (Thr202/Tyr204), MEK1/2 (Ser217/Ser221) and down-stream p90RSK (Ser380). Similarly, PQ10 increases the phosphorylation of β-catenin at Ser552, but showed no effect on phospho-Ser675 levels. After longer durations of treatment, PQ10 reduces β-catenin levels, as well as proteins that are regulated by β-catenin-dependent TCF/LEF-transcriptional activity, such as survivin and cyclin D1. These effects were mimicked by a novel structurally unrelated PDE10 inhibitor, ADT-020 that selectively inhibits the growth of NSCLC cells and increases cGMP levels in a manner similar to PQ10. ADT-020 also activates PKG in NSCLC cells demonstrated by higher phospho-Ser239 VASP levels and lower β-catenin and survivin levels. These observations suggest that PDE10 inhibition and activation of PKG can simultaneously suppress ras and Wnt signaling that are essential for lung tumor cell growth. Supported by NIH grants 1R01CA155638 and 1R01CA131378 (Piazza). Citation Format: Bing Zhu, Kevin Lee, Joshua Canzoneri, Veronica Ramirez-Alcantara, Sara Sigler, Bernard Gary, Ethan Butler, Adam Keeton, Xi Chen, Michael Boyd, Gary Piazza. Phosphodiesterase 10A inhibition suppresses lung tumor cell growth by activating PKG to inhibit ras and Wnt signaling. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4372. doi:10.1158/1538-7445.AM2015-4372

Abstract 950: Antitumor activity of a novel phosphodiesterase 10 inhibitor in an orthotopic mouse model of lung cancer

Abstract BACKGROUND: We recently reported that phosphodiesterase 10A (PDE10) is elevated in certain tumor types and essential for tumor cell growth (Li et al., Oncogene 2014). Here we evaluate anti-tumor efficacy, pharmacokinetics, tissue distribution, and tolerance of a novel PDE10 inhibitor, ADT-048, in mice. METHODS: ADT-048 was orally administrated to C57BL/6 mice and free levels quantified in plasma and tissues by LC-MS. Anti-tumor activity was evaluated in athymic nude-Foxn1nu mice after inoculating the left lung with 1×106 A549 lung tumor cells transfected with luciferase. Tumor growth was monitored by in situ bioluminescence using the In Vivo Imaging System and efficacy was evaluated by necropsy grading. RESULTS: ADT-048 inhibited A549 lung tumor cell growth with an IC50 value of 0.7 μM, while appreciably higher concentrations were required to inhibit the growth of normal airway epithelial cells (IC50 = 35 μM). Pharmacokinetic studies demonstrated a half-life suitable for once a day dosing, while tissue distribution studies demonstrated a unique capacity to achieve high concentrations in lungs relative to plasma and other tissues. ADT-048, at a dose of 100 mg/Kg, displayed significant anti-tumor activity as evident by reduced luminescence and tumor grading following 28 days of treatment. CONCLUSIONS: A novel PDE10 inhibitor, ADT-048, was found to display highly potent and selective tumor cell growth inhibitory activity in vitro. ADT-048 was well tolerated in mice and had attractive pharmaceutical properties with a unique capacity to achieve high lung concentrations following oral administration. Efficacy studies demonstrate strong anti-tumor activity of ADT-048, which represents a candidate for clinical development. Supported by NIH grants 1R01CA155638 and 1R01CA131378 (Piazza). Citation Format: Veronica Ramirez-Alcantara, Michele Schuler, Bing Zhu, Nan Li, Evrim Gurpinar, Dennis Otali, Joshua Canzoneri, Adam Keeton, Bernard Gary, Suzanne Russo, Lori Coward, Gregory Gorman, William Grizzle, Xi Chen, Michael Boyd, Gary Piazza. Antitumor activity of a novel phosphodiesterase 10 inhibitor in an orthotopic mouse model of lung cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 950. doi:10.1158/1538-7445.AM2015-950

Overexpression of microRNA‑125a‑3p effectively inhibits the cell growth and invasion of lung cancer cells by regulating the mouse double minute 2 homolog/p53 signaling pathway.

MicroRNAs (miRs) are a family of small non-coding RNAs that are 21‑24 nucleotides in length. Decreased expression of hsa‑miR‑125a‑3p is observed in a number of patients with non‑small cell lung cancer; however, it is not clear how this miRNA regulates the growth and invasion of lung tumor cells. The aim of the present study was to identify the function of hsa‑miR‑125a‑3p in the growth and invasion of lung cancer cells. The expression of hsa‑miR‑125a‑3p in the A549, NCI‑H460 and SPCA‑1 lung cancer cell lines was analyzed by reverse transcription‑quantitative polymerase chain reaction and the human bronchiolar epithelium cell line (HBE) was used as a control. The results demonstrated that the expression of hsa‑miR‑125a‑3p was significantly lower in NCI‑H460, A549 and SPCA‑1 cells, compared with that in HBE cells. Overexpression of sense miR‑125a‑3p in the A549 lung cancer cell line inhibited cell proliferation for 5‑7 days (P<0.01), and transfection of antisense miR‑125a‑3p did not suppress the cell growth of the lung cancer cells. In addition, overexpression of miR‑125a‑3p in the NCI‑H460 lung cancer cell line markedly induced cell apoptosis, which was detected by fluorescence‑activated cell sorting with annexin V‑fluorescein isothiocyanate/propidium iodide staining. The results of the Transwell migration assay also revealed that transfection of miR‑125a‑3p resulted in decreased migration of lung cancer tumor cells. The pro‑apoptotic gene p53 expression was detected by western blot analysis. The results revealed that the expression of mouse double minute (MDM)‑2 homolog, the principal cellular antagonist of p53, was decreased and p53 expression was upregulated in sense has‑miR‑125a‑3p transfected A549 cells. This was consistent with that observed in NCI‑H460 cells, suggesting that hsa‑miR‑125a‑3p may be involved in the regulation of the MDM2/p53 signaling pathway in lung cancer cells. In conclusion, overexpression of hsa‑miR‑125a‑3p significantly inhibited the proliferation and invasion of lung cancer cells, which may aid in determining the mechanisms underlying the development of lung cancer.

Mitochondrial catalase suppresses naturally occurring lung cancer in old mice.

Lung cancer is generally difficult to detect until the late stages of disease, when it is much more difficult to treat because of the more aggressive and invasive behavior. Advanced lung cancer is much more common in older adults making it even more challenging to treat. Adenocarcinoma belongs to a category of non-small cell lung cancers, which comprise up to 40% of all lung cancers, and about half of these have an activating K-ras mutation. Because treatment relapses are common, more effective unconventional treatment and prevention methods are needed. In this regard, the antioxidant enzyme catalase targeted to mitochondria (mCAT) has been shown to delay aging and cancer in mice, and the progression of transgenic oncogene and syngeneic tumors was suppressed, helping support the notion that attenuation of mitochondria-generated hydrogen peroxide signaling is associated with an antitumor effect. In order to determine if mCAT has any effect on naturally occurring lung cancer of the adenocarcinoma type in old mice, the tumor incidence and progression were examined in the lungs of old mCAT transgenic and wild-type (WT) mice with a CB6F1 (Balb/c X C57BL/6) background. CB6F1 mice with a WT genotype were found to have a high incidence of adenomas at 24 months of age, which progressed to adenocarcinomas at 32 months of age. CB6F1 mice with the mCAT genotype had significantly reduced incidence and severity of lung tumors at both ages. Fibroblasts isolated from the lungs of old mCAT mice, but not WT mice, were shown to secrete soluble factors that inhibited lung tumor cell growth suggesting that stromal fibroblasts play a role in mediating the antitumor effects of mCAT. The aged CB6F1 mouse, with its high incidence of K-ras mutant lung cancer, is an excellent model to further study the anticancer potential of mitochondria-targeted therapy.

Abstract 1761: PDE10, a novel therapeutic target for lung cancer

Abstract Phosphodiesterase 10A (PDE10) is a relatively new PDE isozyme that has been mostly studied in the brain as a therapeutic target for certain psychiatric and neurological conditions, but has not been associated with tumorigenesis. While other investigators have reported low levels of PDE10 in most adult peripheral tissues, we found a large percentage of human lung tumors express high levels of PDE10 compared with normal lung tissue as determined by real-time PCR analysis of cDNA arrays. Statistical analysis revealed that 68% of lung tumors (n=19) displayed more than two-fold increase compared with normal lung tissue, while 26% showed greater than a 10-fold difference. High levels of PDE10 mRNA and protein were also found in human non-small cell lung cancer (NSCLC) cell lines compared with normal airway epithelial cells. PDE10 knockdown by small interference RNA significantly suppressed lung tumor cell growth, as did PDE10 selective inhibitors, papaverine, PQ-10 and Pf-2545920. A novel PDE10 inhibitor, MCI-020 also inhibited the growth of NSCLC cells with a high degree of selectivity for tumor cells. Consistent with a mechanism involving PDE10 inhibition, PQ-10 and MCI-020 increased cGMP levels in a concentration-dependent manner as determined using a cGMP biosensor assay. MCI-020 also activated cGMP-dependent protein kinase (PKG) in NSCLCs as evident by increased phosphorylation of VASP-Serine239. With attractive pharmacokinetic properties, MCI-020 was studied further and found to display an unusual characteristic of achieving high concentrations in lung tissue compared with other tissues and plasma following oral administration. MCI-020 was well tolerated in mice at a dose of 250 mg/kg bid and strongly inhibited tumor formation in an orthotopic model of lung cancer at a dose of 150 mg/kg bid. These observations suggest that PDE10 is essential for lung tumor cell growth and represents a novel therapeutic target for lung cancer. Citation Format: Bing Zhu, Nan Li, Veronica Ramirez-Alcantara, Joshua Canzoneri, Evrim Gurpinar, Alexandra Fajardo, Kevin Lee, Sara Sigler, Bernard Gary, Meagan Thomas, Adam Keeton, Xi Chen, William Grizzle, Gary Piazza. PDE10, a novel therapeutic target for lung cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1761. doi:10.1158/1538-7445.AM2014-1761

Abstract 1768: A novel PDE10 inhibitor suppresses tumor growth in an orthotopic mouse model of lung cancer

Abstract Background: Conventional chemotherapy and radiation cannot distinguish between cancer and normal cells resulting in dose-limiting toxicity. Molecular targeted drugs hold greater promise, but few targets are unique to cancer cells and drugable. Our laboratory discovered that the cyclic nucleotide phosphodiesterase, PDE10, is elevated in human lung tumor cells and essential for their growth. A novel PDE10 inhibitor, MCI-020 was designed to achieve high lung concentrations and found to selectively inhibit the growth of human lung tumor cells in vitro. Objective: Evaluate pharmacokinetics, tissue distribution and antitumor efficacy of MCI-020 in mice. Material and Methods: MCI-020 (100 mg/kg) was formulated as a suspension and administrated orally to C57BL/6 female mice. Free levels of MCI-020 were quantified in plasma and tissues by reverse-phase chromatography with tandem mass spectrometry detection. For efficacy evaluation, athymic nude-Foxn1nu female mice were treated with MCI-020 (150 mg/kg bid) for 5 days before inoculation with 1x106 A549 cells/mouse into the left lung. After inoculation, MCI-020 treatment continued for 4 weeks. Anti-tumor efficacy of MCI-020 was evaluated by gross and histological grading of the lungs. Results: MCI-020 displayed attractive oral bioavailability and reached appreciably higher concentrations in lungs compared with plasma. MCI-020 treatment showed strong anti-tumor efficacy in the orthotopic mouse model of lung cancer and was well tolerated. Compared with the vehicle group in which 85% of the mice formed lung tumors, only 25% of the mice in the treatment group developed tumors. Histological grading of the extent of tumor formation revealed a score of 0.83 ± 0.3 (low involvement) for the treatment group, compared with 2.6 ± 0.4 (high involvement) for the vehicle group. Conclusions: These observations support a critical role of PDE10 in lung tumor growth and suggest that PDE10 inhibitors will be useful for the prevention or treatment of lung cancer. This project is supported by NIH grant 1R01CA148817. Citation Format: Veronica Ramirez-Alcantara, Michele A. Schuler, Bing Zhu, Nan Li, Evrim Gurpinar, Joshua Canzoneri, Adam Keeton, Bernard Gary, Suzanne Russo, Lori Coward, Gregory S. Gorman, William Grizzle, Xi Chen, Gary A. Piazza. A novel PDE10 inhibitor suppresses tumor growth in an orthotopic mouse model of lung cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1768. doi:10.1158/1538-7445.AM2014-1768

Synthesis, characterization, and antitumor activity of unusual pseudo five coordinate gold(III) complexes: Distinct cytotoxic mechanism or expensive ligand delivery systems?

Gold(III) complexes bearing bidentate ligands based on the 1,10-phenanthroline and 2,2′-bipyridine scaffolds have shown promising anticancer activity against a variety of tumor cell lines. In particular, our laboratory has previously found that a pseudo five coordinate gold(III) complex possessing the 2,9-di-sec-butyl-1,10-phenanthroline ligand {[(di-sec-butylphen)AuCl3]} exhibits antitumor activity against a panel of five different lung and head–neck tumor cell lines. However, the [(di-sec-butylphen)AuCl3] complex was determined to be less active than the free 2,9-di-sec-butyl-1,10-phenanthroline ligand. In order to determine if this class of gold(III) complexes has a distinct mechanism of initiating tumor cell death or if these gold complexes simply release the polypyridyl ligand in the intracellular environment, structural analogues of the [(di-sec-butylphen)AuCl3] complex have been synthesized and structurally characterized. These structural congeners were prepared by using mono-alkyl and di-phenyl substituted 1,10-phenanthroline ligands, di-alkyl and di-phenyl substituted 4-methyl-1,10-phenanthroline ligands, and mono-alkyl 2,2′-bipyridine ligands. The redox stability of this library of distorted square pyramidal gold(III) complexes has been studied and the in vitro antitumor activity of gold(III) complexes and corresponding polypyridyl ligands has been determined. The [(di-n-butylphen)AuCl3] and [(mono-n-butylphen)AuCl3] complexes have been found to be significantly more potent at inhibiting the growth of A549 lung tumor cells than the clinically used drug cisplatin. More importantly, these two gold(III) complexes are significantly more active than their respective free ligands, providing evidence that this class of pseudo five coordinate gold(III) complexes has a mechanism of initiating tumor cell death that is independent of the free ligand.

PDE10 inhibitors suppress lung tumor growth in mice.

e22187 Background: Phosphodiesterase 10 has been studied in the brain as a therapeutic target for psychiatric and neurological conditions, but has not been associated with tumorigenesis. Low levels of PDE10 exist in adult peripheral tissues and we found that 68% of lung tumors (n=19) displayed more than 2-fold increase compared with normal lung, and 26% showed greater than a 10-fold difference. High levels of PDE10 mRNA and protein were also found in human NSCLC cells compared with normal airway epithelium. PDE10 knockdown by siRNA suppressed lung tumor cell growth, as did PDE10 selective inhibitors, papaverine, PQ-10 and Pf-2545920. A novel PDE10 inhibitor, MCI-020 was developed from the indene scaffold of the NSAID, sulindac and was found to potently and selectively inhibit the growth of human NSCLC cells. These observations suggest that PDE10 is essential for lung tumor cell growth and represents a novel therapeutic target for lung cancer. We evaluated pharmacokinetics, tissue distribution and antitumo...

SIRT5 desuccinylates and activates SOD1 to eliminate ROS

Cu/Zn superoxide dismutase (SOD1) is a key antioxidant enzyme. Deficiency of SOD1 is associated with various human diseases, including cancer. Here, we report that SOD1 is succinylated and that succinylation decreases its activity. SIRT5 binds to, desuccinylates and activates SOD1. SOD1-mediated ROS reduction is increased when SIRT5 is co-expressed. Furthermore, mutation of the SOD1 succinylation site inhibits the growth of lung tumor cells. These results reveal a novel post-translational regulation of SOD1 by means of succinylation and SIRT5-dependent desuccinylation, which is important for the growth of lung tumor cells.

A Novel Sulindac Derivative Inhibits Lung Adenocarcinoma Cell Growth through Suppression of Akt/mTOR Signaling and Induction of Autophagy

Nonsteroidal anti-inflammatory drugs such as sulindac sulfide have shown promising antineoplastic activity in multiple tumor types, but toxicities resulting from COX inhibition limit their use in cancer therapy. We recently described a N,N-dimethylethyl amine derivative of sulindac sulfide, sulindac sulfide amide (SSA), that does not inhibit COX-1 or -2, yet displays potent tumor cell growth-inhibitory activity. Here, we studied the basis for the growth-inhibitory effects of SSA on human lung adenocarcinoma cell lines. SSA potently inhibited the growth of lung tumor cells with IC50 values of 2 to 5 μmol/L compared with 44 to 52 μmol/L for sulindac sulfide. SSA also suppressed DNA synthesis and caused a G0-G1 cell-cycle arrest. SSA-induced cell death was associated with characteristics of autophagy, but significant caspase activation or PARP cleavage was not observed after treatment at its IC50 value. siRNA knockdown of Atg7 attenuated SSA-induced autophagy and cell death, whereas pan-caspase inhibitor ZVAD was not able to rescue viability. SSA treatment also inhibited Akt/mTOR signaling and the expression of downstream proteins that are regulated by this pathway. Overexpression of a constitutively active form of Akt was able to reduce autophagy markers and confer resistance to SSA-induced cell death. Our findings provide evidence that SSA inhibits lung tumor cell growth by a mechanism involving autophagy induction through the suppression of Akt/mTOR signaling. This unique mechanism of action, along with its increased potency and lack of COX inhibition, supports the development of SSA or related analogs for the prevention and/or treatment of lung cancer.

Abstract 587: A novel non-COX-inhibitory sulindac derivative induces autophagic cell death by inhibiting Akt signaling in lung cancer cells.

Abstract Nonsteroidal anti-inflammatory drugs (NSAIDs) such as sulindac sulfide (SS) have shown promising antineoplastic activity in multiple tumor types, but toxicities resulting from cyclooxygenase (COX) inhibition limit their chronic use for chemoprevention. We recently described a N,N-dimethylethyl amine derivative of SS referred to as sulindac sulfide amide (SSA) that does not inhibit COX-1 or -2, yet displays potent tumor cell growth inhibitory activity. Here, we studied the basis for the growth inhibitory effects of SSA on human lung adenocarcinoma cell lines. SSA potently inhibited the growth of lung tumor cells with IC50 values of 2-5 μM compared with 44-52 μM for SS. SSA also suppressed DNA synthesis and caused a G0/G1 cell cycle arrest more potently than SS. SSA-induced cell death was associated with characteristics of autophagy, including an increase in acidic vesicular organelle content, elevation of the levels of LC3-II, and an increase in autophagic flux. siRNA knockdown of Atg7 attenuated SSA-induced autophagy and cell death. Surface exposure of phosphatidylserine, cleavage of caspases 3 and 7, as well as PARP were detectable with SSA treatment, although the pan-caspase inhibitor, ZVAD was not able to rescue viability. SSA treatment also inhibited Akt/mTOR signaling and the expression of downstream proteins that are regulated by this pathway. Overexpression of a constitutively active form of Akt was able to reduce autophagy markers and confer resistance to SSA-induced cell death. Our findings provide evidence that SSA induces autophagic cell death in lung cancer cell lines by suppressing Akt/mTOR signaling. This unique mechanism of action along with its increased potency and lack of cyclooxygenase inhibition support the development of SSA or related analogs for the prevention and/or treatment of lung cancer. Funding provided by NIH/NCI grants CA131378 and CA148817. Citation Format: Evrim Gurpinar, William E. Grizzle, John J. Shacka, Nan Li, Adam B. Keeton, Gary A. Piazza. A novel non-COX-inhibitory sulindac derivative induces autophagic cell death by inhibiting Akt signaling in lung cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 587. doi:10.1158/1538-7445.AM2013-587

Abstract LB-355: A novel non-COX-inhibitory sulindac derivative induces autophagic cell death in lung adenocarcinoma cells

Abstract Autophagy is a normal catabolic process, which serves to remove damaged organelles and long-lived proteins within the cell. Several studies have provided evidence that some tumor cell lines respond to chemotherapeutic agents by triggering autophagy and inducing autophagic cell death (or programmed cell death type II) characterized by the formation of autophagic vacuoles in the cytoplasm. This, along with observations that autophagic activity and signaling are reduced during malignant transformation suggest that induction of autophagic cell death may have a therapeutic value in cancer. In this study, we describe a novel non-COX inhibitory derivative of NSAID sulindac, referred to as sulindac sulfide amide (SSA), which was found to inhibit the growth of lung adenocarcinoma cell lines primarily through the induction of autophagy with or without apoptosis. SSA inhibited the growth of human lung tumor cells (A549, H1299, HOP-62) in a dose-dependent manner with IC50 values of 3-5 µM, while sulindac sulfide inhibited growth with IC50 values of 50-60 µM. In addition, SSA treatment led to a G0/G1 arrest and decreased proliferation as measured by EdU incorporation. SSA induced apoptosis of A549 lung tumor cells as determined by caspase 3/7 and AnnexinV assays, however, caspase-induction was absent in H1299 cells suggesting an alternative mechanism of cell death. Interestingly, staining SSA-treated A549 cells with Lyso-ID green dye revealed a dramatic increase in the number and size of acidic vesicles within the cytoplasm. Moreover, histological analysis of dying cells demonstrated multiple, large intracellular vacuoles consistent with autophagy. Western blotting for autophagosome-associated LC3 protein demonstrated that SSA induces a dose- and time-dependent increase in autophagic vacuole formation. This was confirmed by transiently transfecting A549 cells to overexpress the LC3 protein fused to the enhanced green fluorescent protein. 5μM SSA treatment for 24h significantly increased the number of cells with LC3-positive vesicles as well as the number of vesicles within the cytoplasm. These effects were not observed with sulindac sulfide, which suggests a distinct mechanism of action. SSA inhibited the phosphorylation of Akt and its downstream effectors mTOR, 4EBP and p70S6K resulting in induction of autophagy. The expression of AMPK, an inhibitor of mTOR function, was shown to be upregulated while levels of cyclinD1 and survivin, important mediators of tumor cell proliferation and death respectively, were downregulated. Our results suggest that SSA can induce autophagy by a mechanism involving the suppression of Akt/mTor signaling. We conclude that SSA is a highly promising chemopreventive or chemotherapeutic agent due to its ability to induce both caspase-dependent apoptotic and caspase-independent autophagic cell death. Funding provided by NIH/NCI grants CA131378 and CA148817. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-355. doi:1538-7445.AM2012-LB-355

Stimulation of neoplastic mouse lung cell proliferation by alveolar macrophage-derived, insulin-like growth factor-1 can be blocked by inhibiting MEK and PI3K activation

BackgroundWorldwide, lung cancer kills more people than breast, colon and prostate cancer combined. Alterations in macrophage number and function during lung tumorigenesis suggest that these immune effector cells stimulate lung cancer growth. Evidence from cancer models in other tissues suggests that cancer cells actively recruit growth factor-producing macrophages through a reciprocal signaling pathway. While the levels of lung macrophages increase during tumor progression in mouse models of lung cancer, and high pulmonary macrophage content correlates with a poor prognosis in human non-small cell lung cancer, the specific role of alveolar macrophages in lung tumorigenesis is not clear.MethodsAfter culturing either an immortalized lung macrophage cell line or primary murine alveolar macrophages from naïve and lung-tumor bearing mice with primary tumor isolates and immortalized cell lines, the effects on epithelial proliferation and cellular kinase activation were determined. Insulin-like growth factor-1 (IGF-1) was quantified by ELISA, and macrophage conditioned media IGF-1 levels manipulated by IL-4 treatment, immuno-depletion and siRNA transfection.ResultsPrimary macrophages from both naïve and lung-tumor bearing mice stimulated epithelial cell proliferation. The lungs of tumor-bearing mice contained 3.5-times more IGF-1 than naïve littermates, and media conditioned by freshly isolated tumor-educated macrophages contained more IGF-1 than media conditioned by naïve macrophages; IL-4 stimulated IGF-1 production by both macrophage subsets. The ability of macrophage conditioned media to stimulate neoplastic proliferation correlated with media IGF-1 levels, and recombinant IGF-1 alone was sufficient to induce epithelial proliferation in all cell lines evaluated. Macrophage-conditioned media and IGF-1 stimulated lung tumor cell growth in an additive manner, while EGF had no effect. Macrophage-derived factors increased p-Erk1/2, p-Akt and cyclin D1 levels in neoplastic cells, and the combined inhibition of both MEK and PI3K ablated macrophage-mediated increases in epithelial growth.ConclusionsMacrophages produce IGF-1 which directly stimulates neoplastic proliferation through Erk and Akt activation. This observation suggests that combining macrophage ablation therapy with IGF-1R, MEK and/or PI3K inhibition could improve therapeutic response in human lung cancer. Exploring macrophage-based intervention could be a fruitful avenue for future research.

Dietary administration of berberine or Phellodendron amurense extract inhibits cell cycle progression and lung tumorigenesis

Phellodendron amurense extract is a Chinese herbal remedy that has recently been studied for its antitumor, antimicrobial and other biological activities. It is previously unknown if these agents are bioavailable and effective against tumors when delivered as a dietary component. It is also unknown if the anti-tumorigenic properties of berberine, an isoquinoline alkaloid component of P. amurense, is equally effective when administered alone. There are contrasting reports on the cellular processes involved in anti-tumorigenesis by P. amurense and berberine. Here we find that berberine, when administered orally through the diet, inhibits in vivo tumorigenesis of both p53 expressing and p53 null lung tumor xenografts equally whether administered in its pure form or as a part of P. amurense extract. We also show that berberine induces G1 cell cycle arrest, inhibits proliferative kinase signaling and arrests the growth of lung tumor cells in culture. Berberine administered in the diet was detectable by HPLC in the lungs of mice fed P. amurense or equivalent doses of berberine at concentrations of 455 and 518 ng/ml respectively and inhibited the growth of xenografted A549 cell tumors, which grew to 9.4 and 6.4 mm³ respectively, compared to 58.9 mm³ in control mice (P < 0.001). Phosphorylation of Akt, CREB and MAPK was inhibited in A549 cells by P. amurense. Demonstration of oral bioavailability and anti-tumorigenic efficacy of dietary berberine, as well as further demonstration of signaling pathway modulation and cell-cycle arrest, implicate this relatively safe, natural compound as a potentially important therapeutic and chemopreventive agent for lung cancer.

Abstract 2349: DLC1 interaction with S100A10 mediates inhibition of in vitro cell invasion and tumorigenicity of lung cancer cells

Abstract Deleted in liver cancer 1 (DLC1) is a multidomain Rho-GTPase-activating protein (RhoGAP) and a tumor suppressor in several human common cancers. DLC1's structure predicts its interaction with a number of other proteins with various cellular functions. Previously we have shown that S100A10 (also known as p11), a key plasminogen receptor on the surface of cell membrane, colocalizes and binds with DLC1 in cell cytoplasm. The binding sites were mapped to the central domain of the DLC1 and at the end of C-terminus of S100A10. The same terminus is also a binding site for Annexin A2, which is believed to regulate availability of extracellular S100A10 and, therefore, dynamics of plasmin formation. Now we demonstrate that DLC1 competes with Annexin A2 for interaction with S100A10. DLC1 binding to S100A10 did not affect DLC1's RhoGAP activity but did reduce availability of S100A10 for binding by Annexin A2. Furthermore, the expression of DLC1 also affected S100A10 expression in a dose dependent manner, and increased S100A10 ubiquitin-dependent degradation. All of the above interactions attenuated plasminogen activation and resulted in inhibition of in vitro cell migration, invasion, colony formation and anchorage independent growth of aggressive and tumorigenic lung tumor cells. Transduction of DLC1-negative breast tumor cells significantly reduced the ability of tumor cells to form pulmonary metastases as well as inhibited distant dissemination of liver tumor cells in athymic mice. Current observations provide a plausible mechanism for DLC1 antimetastatic function by negatively affecting plasminogen activation, which in turn lead to a significant reduction in invasiveness of tumor cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2349.

Involvement of Akt in synergistic effects of thermo-chemotherapy on human small cell lung cancer H446 cell apoptosis

Akt pathway plays an important role in cell growth and apoptosis. This study was to characterize the role of Akt in the synergistic effects of thermo-chemotherapy on lung cancer cell growth and its underlying mechanisms. H446 cells were subjected to different thermo-chemotherapy schemes: 43centigrade + paclitaxel (120 microg/L) (thermo-chemotherapy group), 43centigrade + paclitaxel (120 microg/L) + Wortmannin (1 micromol/L, PI3K/Akt pathway inhibitor) (Wortmannin group), 43centigrade + paclitaxel (120 microg/L) + N-acety-L-cysteine (NAC) (30 micromol/L, reactive oxygen species, ROS inhibitor) (NAC group), and paclitaxel (120 microg/L) group. The cells without any treatment were used as the control. MTT assay was conducted to measure the cell proliferation rate. Cell apoptosis was analyzed by flow cytometry (FCM). ROS was detected with fluorescence. Phosphorylation of Akt and the expressions of Caspase-3 were determined by Western blot. The cell proliferation rate was significantly lower in the thermo-chemotherapy group than in the control and the chemotherapy groups ((59.83 +/- 3.36)% vs. (100.00 +/- 0.00)% and (69.16 +/- 2.95)%, P < 0.05). The rate of cell apoptosis was the highest in the thermo-chemotherapy group (27.59 +/- 5.47)% (P < 0.05). The ROS expression level was higher in the cells of thermo-chemotherapy group (102.14 +/- 18.34) than in the other groups (P < 0.05), which could be inhibited by NAC(28.01 +/- 1.19), but not by the PI3K inhibitor Wortmannin (99.87 +/- 8.35). Phosphorylation of Akt significantly decreased in the thermo-chemotherapy group (0.69+/-0.03) (P < 0.05), which could be blocked by Wortmannin (0.00 +/- 0.00), but increased by NAC (1.05 +/- 0.29) (P < 0.05). The expression level of Caspase-3 was higher in the thermo-chemotherapy group (1.07 +/- 0.08) than in other groups (P < 0.05). Thermo-chemotherapy has a stronger inhibitory effect than chemotherapy alone in lung tumor cell growth, probably through induction of ROS production and subsequent inhibition of Akt pathway activation and Caspase pathway-induced cancer cell apoptosis.

Specific Accumulation and Antitumor Effects of Hybrid Liposomes on the Growth of Lung Tumor Cells

In general, chemotherapeutic effects were low for non-small cell lung cancer (NSCLC) in the lung tumor. We examined the accumulation and antitumor effects of hybrid liposomes (HL-23) composed of phospholipid (L-α-dimyristoylphosphatidylcholine: DMPC) and PEG surfactant [polyoxyethylene(23)dodecyl ether: C₁₂(EO)₂₃] on NSCLC cells in vitro. Accumulation of HL-23 including a fluorescence probe [1-Palmitoyl-2-[12(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]dodecanoyl]-sn-Glycero-3-Phosphocholine: NBDPC] was observed for NSCLC cells using a confocal laser microscope, but no accumulation of HL-23 in normal lung cells was observed. Furthermore, inhibitory effects of HL-23 on the growth of NSCLC cells were obtained on the basis of a WST-1 assay. It was also clarified that HL-23 induced apoptosis for NSCLC cells on the basis of Annexin-V binding and TUNEL assay. These results suggest that HL-23 could be applied in effective chemotherapies for NSCLC.

Suppression of PMA-induced tumor cell invasion and metastasis by aqueous extract isolated from Prunella vulgaris via the inhibition of NF-κB-dependent MMP-9 expression

Matrix metalloproteinase-9 (MMP-9) plays an important role in the invasion and metastasis of cancer cells. In this study, we examined the inhibitory effects of tumor cell migration and invasion by aqueous extract isolated from Prunella vulgaris (PVAE) using in vitro and in vivo assays. PVAE reduced PMA-induced activation of MMP-9 and further inhibited cell invasion and migration. PVAE suppressed PMA-enhanced expression of MMP-9 protein, mRNA and transcription activity levels through suppression of NF-κB activation without changing the tissue inhibitor of metalloproteinase level. PVAE inhibited PMA-induced NF-κB nuclear translocation, which is upstream of PMA-induced MMP-9 expression and invasion. Furthermore, pretreatment with NF-κB activation inhibitor inhibited the PMA-induced MMP-9 expression and activity. PVAE repressed the PMA-induced phosphorylation of ERK1/2, which is upstream signaling molecules in MMP-9 expression. We confirmed that the inhibitory effect of PVAE on lung metastasis and tumor cell growth using B16-F10 melanoma cells or B16-F1 melanoma cells in C57BL/6 mice. The oral administrations of PVAE reduced the lung metastasis and tumor cell growth by B16-F10 or B16-F1 melanoma cells. These results suggested that the anti-metastatic effect of PVAE is mediated through the suppression of MMP-9 expression by the inhibition of NF-κB via ERK1/2 signaling pathway as well as MMP-9 activity.

Recombinant Human IL-24 Suppresses Lung Carcinoma Cell Growth via Induction of Cell Apoptosis and Inhibition of Tumor Angiogenesis

Previous studies have shown that interleukin-24 (IL-24; mda-7) as a novel tumor suppressor gene has tumor-suppressive activity against a broad spectrum of human cancers. However, the therapeutic effect of the recombinant human IL-24 (rhIL-24) protein purified from prokaryotic cells on human lung cancers has not been reported. In this study, we cloned the human gene coding for IL-24 from lipopolysaccharide-activated human peripheral blood mononuclear cells (PBMCs) by reverse-transcriptase polymerase chain reaction and constructed an expression vector pBV220-IL-24. We then transfected Escherichia coli DH5alpha with pBV220-IL-24. The soluble rhIL-24 was obtained from purified insoluble inclusion bodies of transfected cells by a denaturing and renaturing process. We demonstrated that the purified soluble rhIL-24 protein with 18.5 kappaDa was capable of (1) inducing in vitro apoptosis of A549 lung carcinoma cells; (2) activating PBMCs to secrete cytokines such as IL-6, tumor necrosis factor-alpha, and interferon-gamma; (3) inhibiting the formation of blood capillaries on chicken embryonic allantois and in vivo tumor angiogenesis; and (4) inhibiting A549 lung tumor cell growth in vitro and in vivo. Therefore, our results indicate its potent suppressive effect on human lung carcinoma cell line and warrant its further investigation for therapeutic application against human lung cancer.

Antitumor and Immunostimulatory Effects of Residual Powder from Barley-Shochu Distillation Remnants

The development of effective utilization methods of Shochu distillation remnants is desirable from the viewpoint of environmental protection and biomass. In this study, we examined inhibitory effects of the powder (barleypowder) obtained from the barley-Shochu distillation remnants on the various tumor cells in vitro and immunostimulation effect in vivo .R emarkable inhibitory effects on the growth of human lung tumor cells along with the induction of apoptosis were obtained for barley-powder in vitro .O nt he other hand, the barley-powder induced the production of serum Interferon (IFN)-γ for normal rats in vivo ,a nd increased natural killer (NK) activity. Furthermore, the barley-powder showed no acute toxicity in the tail vein administration to normal rats. It was found that the barley-powder had not only an antitumor effect but also an immunostimulation effect for the first time.