Non-small Cell Lung Cancer H460 Research Articles

Abstract 440: The sublethal dose radiation induces cellular senescence and potent bystander effects through c-Myc oncogene.

Abstract The sublethal dose radiation may induce cellular senescence and promote secondary maligancy during radiotherapy. However, the molecular mechanisms are unclear. It has been reported that a variety of early response oncogenes can be up-regulated by the low dose radiation. Because oncogene-induced senescence (OIS) has been considered important for tumor development, we are interested in investigating whether the sublethal dose radiation also raises OIS to affect the malignancy. To this end, human diploid fibroblasts WI-38 cells and human non-small lung cancer H1299 cells were exposed to less than 2Gy of X-rays delivered by the RS2000 X-rays machine. Both cell types exhibited senescent phenotypes within a dose-dependent manner using the senescence-associated β-galactosidase (SA-β-gal) staining. Subsequently, we investigated the expression of c-Myc and c-Ras oncogenes, and the results showed that sublethal dose radiation could induce c-Myc, but not c-Ras, in both transcriptional and translational levels. Additionally, up-regulated c-Myc exhibited transcriptional activity as demonstrated by the promoter-based reporter gene assys. Although the sublethal dose radiation caused cellular senescence and growth delay in exposed cells, the collected culture medium could increase the motility of non-irradiated H1299 cancer cells using the wound healing assay. Interestingly, the culture medium obtained from irradiated H1299 cancer cells caused higher motility in non-irradiated cells than that from irradiated WI-38 fibroblasts. Furthermore, the sublethal dose radiation induced senescent phenotype was suppressed in cells treated with siRNA against c-Myc. Meanwhile, the motility of non-irradiated cells was not increased by the cultured medium obtained from irradiated c-Myc silenced cells. Taken together, the current data suggest that the sublethal dose radiation causes a c-Myc oncogene induced senescence, and this event may promote the bystandard effects that influence the motility of non-irradiated cancer cells. Citation Format: Chia-Chien Lo, Yen-Ting Chou, Su-Jun Chiu, Jeng-Jung Hwang, Yi-Jang Lee. The sublethal dose radiation induces cellular senescence and potent bystander effects through c-Myc oncogene. [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 440. doi:10.1158/1538-7445.AM2013-440 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Abstract 1309: ERβ1 induces apoptosis in lung cancer cells by regulating growth factor signaling.

Abstract Estrogens have recently been associated with the clinical outcome of patients with non-small cell lung cancer (NSCLC). The wild-type estrogen receptor β (ERβ1) one of the mediators of the estrogen signaling in cells is expressed in lung cancer. However, the role of ERβ1 in NSCLC cell growth has not been fully elucidated. Here we show that induction of ERβ1 expression inhibited cell growth in H1299 and A549 NSCLC cells. Treatment of these cells with the ERβ agonists 17β-estradiol or 3β-Adiol further decreased cell proliferation and induced apoptosis. ERβ1 was found to induce apoptosis through stimulation of the intrinsic apoptotic pathway that involves upregulation of the proapoptotic protein Bim. Inactivation of the extracellular-signal-regulated kinases ERK1/2 was found to be involved in the ERβ1-mediated induction of apoptosis since treatment with EGF that activates ERK1/2 abolished the ability of ERβ1 to sustain the apoptotic phenotype. These results suggest that, by regulating NSCLC cell growth, ERβ1 could be an important factor in biology of lung cancer and a potentially useful component in the clinical management of the disease. Citation Format: Christoforos Thomas, Fotis Nikolos, Gayani Rajapaksa, Igor Bado, Jan-Ake Gustafsson. ERβ1 induces apoptosis in lung cancer cells by regulating growth factor signaling. [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 1309. doi:10.1158/1538-7445.AM2013-1309

Abstract 4530: Nanoparticle formulation of KU55933 as a potent radiosensitizer.

Abstract KU55933 is a potent and specific inhibitor of the Ataxia Telangiectasia Mutated (ATM) protein. Given ATM's critical role in DNA damage repair, specifically double-strand break repair, KU55933 holds high potential as a radiosensitizer. Indeed, previous preclinical studies have demonstrated the radiosentization effect of KU55933. However, clinical translation of KU55933 as a radiosensitizer has been prevented due to concerns of its potential toxicity. Systemic administration of KU55933 would sensitize normal cells as well as cancer cells to the effects of radiotherapy. Such sensitization would not improve the therapeutic index of radiotherapy. Therefore, the key challenge in the translation of KU55933 is to identify methods that can selectively deliver KU55933 to tumors while minimizing normal tissue dose. While traditional drug delivery methods cannot overcome this challenge, the development of nanoparticle (NP) drug delivery vehicles offers a unique opportunity. NPs are known to preferentially accumulate in tumors while provide low drug dose to nearby normal tissues. We hypothesize that NP delivery of KU55933 can not only improve the therapeutic efficacy of KU55933, but also lower its toxicity. In this study, we engineered a NP formulation of KU55933 using a biodegradable lipid-polymer NP. We then compared the radiosensitization effect of NP KU55933 to that of KU55933 in vitro using the non-small cell lung cancer (NSCLC) cell lines H23, A549 and H460. Clonogenic survival assays showed that NP KU55933 is a more potent radiosensitizer than KU55933. The differential radiosensitization effect of both KU55933 formulations was also evaluated in a xenograft mouse model of NSCLC. We validated our in vitro results by demonstrating that NP KU55933 is more effective than KU55933 as radiosensitizer in flank xenograft models of NSCLC. To compare the toxicity of NP KU55933 and KU55933, we determined the skin toxicity after radiotherapy with the two drug formulations. We found lower skin toxicity in mice treated with the NP KU55933 than that for KU55933. In conclusion, our studies demonstrate that NP formulation of KU55933 can enhance the radiosensitization effect and decreases the toxicity in vitro and in vivo. Our results suggest that NP KU55933 holds potential for clinical translation. Citation Format: Haydee Lara, Michael B. Foote, Edina C. Wang, Manish Sethi, Patricia Casbas-Hernandez, Andrew Z. Wang. Nanoparticle formulation of KU55933 as a potent radiosensitizer. [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 4530. doi:10.1158/1538-7445.AM2013-4530

Abstract 5656: Sustained delivery of CZ48, a lactone-stabilized camptothecin, by nanosuspensions.

Abstract Background: CZ48, a prodrug of camptothecin (CPT), shows promising antitumor activity as a topoisomerase I (Topo-I) inhibitor after its biotransformation to CPT in vivo. Studies establish that a prolonged exposure of Topo-I inhibitors with low concentrations is more efficacious in cancer treatment than a short term exposure with high concentrations, due to the S-phase cell cycle specific mechanism. However, poor solubility and the effective delivery challenges have been the obstacles for clinical applications of CZ48 to patients. Objectives: The research was to achieve the following objectives: (1) to prepare by wet media milling method and characterize CZ48 nanosuspensions (NSs); (2) to determine the pharmacokinetics (PK) of CZ48 and CPT from CZ48 NSs in Swiss nude mouse model; (3) to demonstrate the proof-of-concept efficacy of CZ48 NS in tumor-bearing athymic mouse model. Methods: CZ48 NSs of different sizes were formulated by media milling method. The in vitro release kinetics in plasma and phosphate buffered saline (PBS) solution (pH 7.4) were characterized, respectively, using the dialysis bag diffusion technique. The PK of CZ48 and CPT were evaluated in male Swiss nu-nu mice by intravenous (i.v.) doses of 5 and 25 mg/kg for cosolvent reference and NSs, respectively. The PK parameters of CZ48 and CPT were derived by WinNonlin 3.0. The proof-of-concept efficacy was performed in the subcutaneous tumor model of Non-small Cell Lung Cancer (NSCLC) H460 cell line in the nude mouse model. ANOVA with Tukey's post-hoc and Kaplan-Meier survival analysis were used for comparisons of PK modifications and efficacy of the CZ48 from NSs using the cosolvent as reference. Results: CZ48 NSs of particle sizes of 200 nm (NS-S) and 600 nm (NS-L) were successfully formulated and extensively characterized. The in vitro sustained release of CZ48 from NSs in plasma was achieved with 0.60 ± 0.12 %/h release rate and less than 40 % total release at 48 h, compared with 6.48 ± 0.29 %/h release rate and complete release by 28 h from cosolvent. NS-S and NS-L prolonged the blood circulation of CZ48 by 8.00, and 5.58 h, respectively, compare to 0.70 h from cosolvent. More importantly, NS-S yielded CPT with the longest half-life (12.51, 5.81 vs. 0.40 h) and largest AUC (19.78, 10.95 vs. 8.29 nM*h/(mg/kg)) than NS-L and cosolvent. CZ48-loaded NS-S exhibited significant tumor growth rate suppression compared to cosolvent (0.08 ± 0.03 vs. 0.18 ± 0.06/day) with a higher tolerable dose (25 vs. 5 mg/kg), and an improved survival rate (80% vs. 10%). Conclusion: This study is the first report to demonstrate the NS as a viable pharmaceutical carrier to deliver CZ48 in a sustained manner, achieving significant tumor growth suppression at a higher tolerable dose. The study provides a formulation strategy of great potentials to overcome the delivery barriers for other anticancer drugs. We anticipate that the CZ48 NS will be a lead candidate for the future clinical trials of CZ48. Citation Format: Diana S-L Chow, Dong Dong, Beppino C. Giovanella, Xin Liu, Cheng-Hui Hsiao, Yang Teng. Sustained delivery of CZ48, a lactone-stabilized camptothecin, by nanosuspensions. [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 5656. doi:10.1158/1538-7445.AM2013-5656

Abstract 1022: Carfilzomib demonstrates broad antitumor activity in preclinical lung cancer models.

Abstract Carfilzomib (CFZ), is an irreversible inhibitor of the chymotrypsin-like activity of the proteasome that was recently FDA approved for treatment of refractory and relapsed multiple myeloma. Early Phase 1B studies of CFZ showed signs of clinical activity in a number of solid tumors, including non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC). We investigated the anti-tumor activity of CFZ monotherapy and in combination with cisplatin in NSCLC and SCLC cell lines and xenograft models. CFZ showed marked anti-proliferative activity in A549, H1993, H520, H460, H1299 NSCLC cell lines, with IC50 values after 96 hours of treatment ranging from 1.0 nM to 27 nM. CFZ + cisplatin resulted in antagonistic anti-proliferative activity in the NSCLC A549, H1993, and H520 cells, independent of sequence of drug administration. In vivo, CFZ monotherapy resulted in tumor growth inhibition in an A549 NSCLC murine xenograft model. In contrast, the SCLC cell lines SHP77 and H69 were more resistant to the anti-proliferative effects of CFZ, with IC50 values at 96 hours of 0.917 μM and >30 μM, respectively. In vitro, in the SHP-77 SCLC cell line CFZ + Cisplatin showed synergistic activity. In vivo, in a SHP-77 SCLC SCID murine xenograft model CFZ monotherapy resulted in an 8-day tumor growth delay to reach 1500 mm3. No additive or synergistic anti-tumor efficacy was observed for CFZ + cisplatin in this model. Western blot analysis from SHP-77 cell lines grown in vitro and treated with CFZ showed an increase in cleaved PARP and p53, supporting activation of apoptosis. We will report on inhibition of the chymotrypsin-like proteasome activity in CFZ-treated lung cancer cell lines. Together, these pre-clinical studies support further preclinical and clinical investigations of CFZ in NSCLC and SCLC, including novel CFZ + anti-cancer drug combinations. Citation Format: Amanda F. Baker, Barbara Sands, Lilliana Carbajal, Janet Anderl, Elena T. Chan, Christopher J. Kirk, Linda L. Garland. Carfilzomib demonstrates broad antitumor activity in preclinical lung cancer models. [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 1022. doi:10.1158/1538-7445.AM2013-1022

Abstract 5650: Determinants of acquired resistance to PI3K/mTOR inhibition in cells refractory to EGFR inhibition.

Abstract Acquired resistance to EGFR inhibitors can be mediated through PI3K/mTOR pathway activation. This has prompted the consideration of PI3K/mTOR inhibitors for treatment of EGFR inhibitor-refractory non-small cell lung cancer (NSCLC). This study aimed to generate and elucidate mechanisms of acquired resistance to BEZ235, a PI3K/mTOR inhibitor, in H1975 NSCLC cells containing the EGFR T790M resistance mutant. IC50 values of compounds were determined at 72 hours using the MTS assay. Acquired resistant clones of H1975 were generated by continual exposure of H1975 cells to parental IC50 concentrations of BEZ235, followed by monoclonal selection. Protein levels, cell invasion/migration, and cell cycle analysis were evaluated using standard techniques. Apoptosis was measured using Roche Cell Death kit. Two resistant clones were generated with stably higher IC50 values (Clone 5 2.29uM, Clone 6 1.94uM) to parental H1975 cells (0.32uM) after 6 months. The clones were resistant to other PI3K/mTOR inhibitors but not other cytotoxic compounds or targeted agents such as EGFR/MEK inhibitors. Following BEZ235 treatment, acquired resistant clones underwent less cell cycle G1 arrest and displayed greater invasion and migration compared to parental cells. No significant differences were seen in basal levels of pRTKs, pAKT, pmTOR, pS6 and p4EBP1, and apoptosis. In summary, EGFR inhibitor-refractory cells with acquired resistance to PI3K/mTOR inhibition have been generated and characterized with phenotypes suggestive of a class-mediated mechanism of acquired resistance. Possible mediators of acquired resistance have emerged following RNA-sequencing and reverse phase protein array analyses and are currently being validated. Citation Format: King Xin Koh, Bhaskar Bhattacharya, Joanne Loh, Hong Kiat Ng, Ross Soo, Barry Iacopetta, Richie Soong. Determinants of acquired resistance to PI3K/mTOR inhibition in cells refractory to EGFR inhibition. [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 5650. doi:10.1158/1538-7445.AM2013-5650

Honokiol inhibits non-small cell lung cancer cell migration by targeting PGE₂-mediated activation of β-catenin signaling.

Lung cancer remains a leading cause of death due to its metastasis to distant organs. We have examined the effect of honokiol, a bioactive constituent from the Magnolia plant, on human non-small cell lung cancer (NSCLC) cell migration and the molecular mechanisms underlying this effect. Using an in vitro cell migration assay, we found that treatment of A549, H1299, H460 and H226 NSCLC cells with honokiol resulted in inhibition of migration of these cells in a dose-dependent manner, which was associated with a reduction in the levels of cyclooxygenase-2 (COX-2) and prostaglandin E2 (PGE2). Celecoxib, a COX-2 inhibitor, also inhibited cell migration. Honokiol inhibited PGE2-enhanced migration of NSCLC cells, inhibited the activation of NF-κB/p65, an upstream regulator of COX-2, in A549 and H1299 cells, and treatment of cells with caffeic acid phenethyl ester, an inhibitor of NF-κB, also inhibited migration of NSCLC cells. PGE2 has been shown to activate β-catenin signaling, which contributes to cancer cell migration. Therefore, we checked the effect of honokiol on β-catenin signaling. It was observed that treatment of NSCLC cells with honokiol degraded cytosolic β-catenin, reduced nuclear accumulation of β-catenin and down-regulated matrix metalloproteinase (MMP)-2 and MMP-9, which are the down-stream targets of β-catenin and play a crucial role in cancer cell metastasis. Honokiol enhanced: (i) the levels of casein kinase-1α, glycogen synthase kinase-3β, and (ii) phosphorylation of β-catenin on critical residues Ser45, Ser33/37 and Thr41. These events play important roles in degradation or inactivation of β-catenin. Treatment of celecoxib also reduced nuclear accumulation of β-catenin in NSCLC cells. FH535, an inhibitor of Wnt/β-catenin pathway, inhibited PGE2-enhanced cell migration of A549 and H1299 cells. These results indicate that honokiol inhibits non-small cell lung cancer cells migration by targeting PGE2-mediated activation of β-catenin signaling.

Global Decrease of Histone H3K27 Acetylation in ZEB1-Induced Epithelial to Mesenchymal Transition in Lung Cancer Cells

The epithelial to mesenchymal transition (EMT) enables epithelial cells with a migratory mesenchymal phenotype. It is activated in cancer cells and is involved in invasion, metastasis and stem-like properties. ZEB1, an E-box binding transcription factor, is a major suppressor of epithelial genes in lung cancer. In the present study, we show that in H358 non-small cell lung cancer cells, ZEB1 downregulates EpCAM (coding for an epithelial cell adhesion molecule), ESRP1 (epithelial splicing regulatory protein), ST14 (a membrane associated serine protease involved in HGF processing) and RAB25 (a small G-protein) by direct binding to these genes. Following ZEB1 induction, acetylation of histone H4 and histone H3 on lysine 9 (H3K9) and 27 (H3K27) was decreased on ZEB1 binding sites on these genes as demonstrated by chromatin immunoprecipitation. Of note, decreased H3K27 acetylation could be also detected by western blot and immunocytochemistry in ZEB1 induced cells. In lung cancers, H3K27 acetylation level was higher in the tumor compartment than in the corresponding stroma where ZEB1 was more often expressed. Since HDAC and DNA methylation inhibitors increased expression of ZEB1 target genes, targeting these epigenetic modifications would be expected to reduce metastasis.

Enhanced cellular radiosensitivity induced by cofilin-1 over-expression is associated with reduced DNA repair capacity

Purpose: A previous report has indicated that over-expression of cofilin-1 (CFL-1), a member of the actin depolymerizing factor (ADF)/cofilin protein family, enhances cellular radiosensitivity. This study explores the involvement of various DNA damage responses and repair systems in the enhanced cellular radiosensitivity as well as assessing the role of CFL-1 phosphorylation in radiosensitivity.Materials and methods: Human non-small lung cancer H1299 cells harboring a tet-on gene expression system were used to induce exogenous expression of wild-type CFL-1. Colony formation assays were used to determine cell survival after γ-ray exposure. DNA damage levels were determined by Comet assay. DNA repair capacity was assessed by fluorescence-based DNA repair analysis and antibody detection of various repair proteins. The effects of CFL-1 phosphorylation on radiation responses were explored using two mutant CFL-1 proteins, S3D and S3A. Finally, endogenous CFL-1 phosphorylation levels were investigated using latrunculin A (LA), cytochalasin B (CB) and Y27632.Results: When phosphorylatable CFL-1 was expressed, radiosensitivity was enhanced after exposure to γ-rays and this was accompanied by DNA damage. Phosphorylated histone H2AX (γ-H2AX) and p53-binding protein-1 (53BP1) foci, as well as Chk1/2 phosphorylation, were apparently suppressed, although ataxia telangiectasia mutated (ATM) kinase activation was apparently unaffected. In addition, two radiation-induced double-strand break (DSB) repair systems, namely homologous recombination repair (HRR) and non-homologous end joining (NHEJ), were suppressed. Moreover, over-expression of CFL-1 S3D and CFL-1 S3A both enhanced radiosensitivity. However, enhanced radiosensitivity and reduced γ-H2AX expression were only detected in cells treated with LA which increased endogenous phospho-CFL-1, and not in cells treated with Y27632, which dephosphorylates CFL-1.Conclusion: CFL-1 over-expression enhances radiosensitivity and this is associated with reduced DNA repair capacity. Although phosphorylated CFL-1 seems to be involved in radiosensitivity, further studies are required to address the importance of CFL-1 activity to the regulation of radiosensitivity.

MAPK p38 and JNK have opposing activities on TRAIL-induced apoptosis activation in NSCLC H460 cells that involves RIP1 and caspase-8 and is mediated by Mcl-1

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can induce both caspase-dependent apoptosis and kinase activation in tumor cells. Here, we examined the consequences and mechanisms of TRAIL-induced MAPKs p38 and JNK in non-small cell lung cancer (NSCLC) cells. In apoptosis sensitive H460 cells, these kinases were phosphorylated, but not in resistant A549 cells. Time course experiments in H460 cells showed that induction of p38 phosphorylation preceded that of JNK. To explore the function of these kinases in apoptosis activation by TRAIL, chemical inhibitors or siRNAs were employed to impair JNK or p38 functioning. JNK activation counteracted TRAIL-induced apoptosis whereas activation of p38 stimulated apoptosis. Notably, the serine/threonine kinase RIP1 was cleaved following TRAIL treatment, concomitant with detectable JNK phosphorylation. Further examination of the role of RIP1 by short hairpin (sh)RNA-dependent knockdown or inhibition by necrostatin-1 showed that p38 can be phosphorylated in both RIP1-dependent and -independent manner, whereas JNK phosphorylation occurred independent of RIP1. On the other hand JNK appeared to suppress RIP1 cleavage via an unknown mechanism. In addition, only the activation of JNK by TRAIL was caspase-8-dependent. Finally, we identified Mcl-1, a known substrate for p38 and JNK, as a downstream modulator of JNK or p38 activity. Collectively, our data suggest in a subset of NSCLC cells a model in which TRAIL-induced activation of p38 and JNK have counteracting effects on Mcl-1 expression leading to pro- or anti-apoptotic effects, respectively. Strategies aiming to stimulate p38 and inhibit JNK may have benefit for TRAIL-based therapies in NSCLC.

Molecular Imaging of Nonsmall Cell Lung Carcinomas Expressing Active Mutant EGFR Kinase Using PET with [124I]-Morpholino-IPQA

Mutations in the kinase domain of epidermal growth factor receptor (EGFR) have high levels of basal receptor phosphorylation and are associated with clinical responsiveness to Iressa in patients with nonsmall cell lung cancer (NSCLC). This study aimed to assess the feasibility of morpholino-[124I]IPQA derivative as an in vivo PET imaging tool for the expression of different EGFR mutants in NSCLC. In vitro radiotracer accumulation and washout studies demonstrated a rapid accumulation and progressive retention after washout of morpholino-[131I]IPQA derivative in high EGFR-expressing H1299 NSCLC derivative cell lines (L858R and E746-A750 del cell lines), but not in EGFR-transfected H1299 cell line and vector-transfected H1299 cell line. Using the morpholino-[124I]IPQA derivative, we obtained noninvasive microPET images of EGFR activity in L858R and E746-A750 del subcutaneous tumor xenografts, but not in subcutaneous tumor xenografts grown form control cell line. Different EGFR mutant (activity) tumors have a different morpholino-[∗I]IPQA derivative uptake. However, it still needs to modify the structure of IPQA to increase its water solubility and reduce hepatobiliary clearance. Morpholino-[124I]IPQA derivative may be a potential probe for selection of the candidate patients suffering from NSCLC for the small molecule tyrosine kinase inhibitor therapy (e.g., Iressa) in the future.

Acquired differential regulation of caspase-8 in cisplatin-resistant non-small-cell lung cancer

Failure to efficiently induce apoptosis contributes to cisplatin resistance in non-small-cell lung cancer (NSCLC). Although BCL-2-associated X protein (BAX) and BCL-2 antagonist killer (BAK) are critical regulators of the mitochondrial apoptosis pathway, their requirement has not been robustly established in relation to cisplatin. Here, we show that cisplatin can efficiently bypass mitochondrial apoptosis block caused by loss of BAX and BAK, via activation of the extrinsic death receptor pathway in some model cell lines. Apoptosis resistance following cisplatin can only be observed when both extrinsic and intrinsic pathways are blocked, consistent with redundancy between mitochondrial and death receptor pathways in cisplatin-induced apoptosis. In H460 NSCLC cells, caspase-8 cleavage was shown to be induced by cisplatin and is dependent on death receptor 4, death receptor 5, Fas-associated protein with death domain, acid sphingomyelinase and ceramide synthesis. In contrast, cisplatin-resistant cells fail to activate caspase-8 via this pathway despite conserving sensitivity to death ligand-driven activation. Accordingly, caspase-8 activation block acquired during cisplatin resistance, can be bypassed by death receptor agonism.

Combretastatin A-4 derivatives: synthesis and evaluation of 2,4,5-triaryl-1H-imidazoles as potential agents against H1299 (non-small cell lung cancer cell)

A number of 2,4,5-triaryl-1H-imidazole derivatives were synthesized and evaluated for their antiproliferative activities against the growth of five cell lines including three non-small cell lung cancers (H460, H1299, and A549), one breast cancer (MCF-7), and one normal diploid embryonic lung cell line (MRC-5). Preliminary results indicated that both 2-(5-bromofuran-2-yl)-4,5-bis{4-[3-(dimethylamino) propoxy] phenyl}-1H-imidazole (10f) and 4,5-bis{4-[3-(dimethylamino)propoxy]phenyl}-2-(5-nitrofuran-2-yl)-1H -imidazole (10g) were selectively active against the growth of H1229 with an IC(50) of less than 0.1 μM, thus were more active than topotecan (IC(50) > 10.0 μ M). However, both 10f and 10g exhibited only marginal cytotoxicity against H460, A549, MCF-7, and MRC-5 requiring an IC (50) of at least 4.16 μM. Our results also indicated that 10f induced H1299 cell cycle arrest at G0/G1 through the inactivation of p38 MAPK, JNK, ERK, as well as the expression of SIRT1 and survivin. These results suggested that 10f might have therapeutic potential against H1299 (non-small cell lung cancer cell).

EGCG Inhibits Transforming Growth Factor-β-Mediated Epithelial-to-Mesenchymal Transition via the Inhibition of Smad2 and Erk1/2 Signaling Pathways in Nonsmall Cell Lung Cancer Cells

Transforming growth factor-β (TGF-β)-mediated epithelial mesenchymal transition (EMT) of human lung cancer cells may contribute to lung cancer metastasis. It has been reported that EGCG can inhibit tumorigenesis and cancer cell growth in lung cancer; however, the effect of EGCG on EMT in nonsmall cell lung cancer (NSCLC) cells has not been investigated. In this study, we found that NSCLC cells A549 and H1299 were converted to the fibroblastic phenotype in response to TGF-β. Epithelial marker E-cadherin was down-regulated, and mesenchymal marker vimentin was up-regulated simultaneously. Our results illustrated that TGF-β was able to induce EMT in NSCLC cells, and EGCG would reverse TGF-β-induced morphological changes, up-regulate the expression of E-cadherin, and down-regulate the expression of vimentin. Immunofluorescent staining also demonstrated that E-cadherin was up-regulated and that vimentin was down-regulated by EGCG pretreatment. Moreover, wound-healing and the in vitro invasion assay showed that EGCG could inhibit TGF-β-induced migration and invasion of NSCLC cells. By using the dual-luciferase reporter assay, we demonstrated that EGCG inhibited TGF-β-induced EMT at the transcriptional level. EGCG decreased the phosphorylation of Smad2 and Erk1/2, inhibited the nuclear translocation of Smad2, and repressed the expression of transcription factors ZEB1, Snail, Slug, and Twist, and up-regulated the expression of E-cadherin. In summary, our results suggest that EGCG can inhibit TGF-β-induced EMT via down-regulation of phosphorylated Smad2 and Erk1/2 in NSCLC cells.

Lentivirus‐Mediated RNAi Knockdown of NUPR1 Inhibits Human Nonsmall Cell Lung Cancer Growth In Vitro and In Vivo

NUPR1 (nuclear protein 1) was found to play a key role in the development of several malignancies including pancreas, breast, and prostate cancers. However, the functional role of NUPR1 in nonsmall cell lung cancer (NSCLC) progression and development is little known. Here, lentivirus-mediated small interfering RNA (siRNA) was employed to downregulate endogenous NUPR1 expression to study the function of NUPR1 in growth of nonsmall cell lung cancer. A lentivirus-mediated RNAi technology was used to specifically knock down the expression of NUPR1 in H1299 cells. Quantitative real-time reverse transcriptase polymerase chain reaction, flow cytometry, western blot and cell count assays were studied to characterize NUPR1 expression in vitro. Furthermore, nonsmall cell lung cancer xenograft models in nude mice were established to investigate whether knockdown of NUPR1 reduces the tumor growth in vivo. We found that downregulation of NUPR1 expression significantly inhibited nonsmall cell lung cancer H1299 cells proliferation and colony formation in vitro. Moreover, the specific downregulation of NUPR1 arrested cells in G0 phase of cell cycle and increased apoptosis rate. Silencing of NUPR1 also suppressed tumor growth by tail vein injection of lentivirus encoded shRNA against NUPR1 in vivo. Our findings revealed that the NUPR1 gene represents a promising target for gene silencing therapy in nonsmall cell lung cancer.

Erratum to: Flavokawain B induces apotosis of non-small cell lung cancer H460 cells via Bax-initiated mitochondrial and JNK pathway

Erratum to: Flavokawain B induces apotosis of non-small cell lung cancer H460 cells via Bax-initiated mitochondrial and JNK pathway

Targeted delivery of EV peptide to tumor cell cytoplasm using lipid coated calcium carbonate nanoparticles

Intracellular-acting peptide drugs are effective for inhibiting cytoplasmic protein targets, yet face challenges with penetrating the cancer cell membrane. We have developed a lipid nanoparticle formulation that utilizes a pH-sensitive calcium carbonate complexation mechanism to enable the targeted delivery of the intracellular-acting therapeutic peptide EEEEpYFELV (EV) into lung cancer cells. Lipid–calcium-carbonate (LCC) nanoparticles were conjugated with anisamide, a targeting ligand for the sigma receptor which is expressed on lung cancer cells. LCC EV nanoparticle treatment provoked severe apoptotic effects in H460 non-small cell lung cancer cells in vitro. LCC NPs also mediated the specific delivery of Alexa-488-EV peptide to tumor tissue in vivo, provoking a high tumor growth retardation effect with minimal uptake by external organs and no toxic effects.

Mechanisms of antitumor activity by melanoxoin on human non-small cell lung cancer cells

Cancer is a leading cause of death worldwide. The standard treatments of cancer include surgery, chemotherapy, radiation therapy and the newer targeted therapy. Till now, there are various anticancer drugs, which derived from natural products. In this study, we screened a serious of compounds isolated from Pterocarpus santalinus to identify their cell cytotoxicity. We performed the MTT assay to evaluate the cytotoxic effects of test compounds against several human cancer cell lines. Among them, melanoxoin showed the highest cytotoxic effects with a IC50 of 1.98µg/ml in the human non-small cell lung cancer H1299 cells. We next used Human OneArray® v5 to comprehensive analysis the DNA transcription of H1299 cells. It showed that melanoxoin regulated the transcription of several cell cycle regulators in the H1299 cells. Cell cycle analysis by flow cytometry revealed melanoxoin causes G2/M arrest and then increased tumor cell apoptosis. The neutral comet assay revealed that the treatment with melanoxoin induced significant DNA damage in H1299 cells. Therefore, we are interested in the development the molanoxoin as a new source of anti-cancer drug.

Flavokawain B induces apoptosis of non-small cell lung cancer H460 cells via Bax-initiated mitochondrial and JNK pathway

Flavokawain B (FKB) possesses strong anti-neoplastic activity against many cancer cells. Here we assessed its antitumor activity and molecular mechanisms in lung cancer H460 cells in vitro. FKB significantly inhibited cell proliferation and caused arrest of the cell cycle G2-M of H460 cells in a dose-dependent manner. FKB also inducted apoptosis, which was associated with cytochrome c release, caspase-7 and caspase-9 activation and Bcl-xL/Bax dys-regulation. FKB significantly down-regulated survivin and XIAP, and the inhibitory effect induced by FKB was greatly attenuated by through over-expression of survivin or Bax(-/-) MEFs. Furthermore, FKB activated the mitogen-activated protein kinases and the JNK inhibitor SP600125 significantly decreased the growth-inhibitory and apoptotic effects of FKB. Together, these results suggest the anti-lung cancer potential of flavokawain B for the prevention and treatment of lung cancer.

Effect and mechanism of gefitinib inhibition on non-small cell lung cancer radiosensitivity of HCC827 and H358 cell lines

背景与目的表皮生长因子受体（epidermal growth factor receptor, EGFR）是决定放疗效应的一个重要因素，其过表达或是下游通路的激活与包括非小细胞肺癌在内的肿瘤的放疗抵抗相关，因而阻断EGFR的信号通路可能会增强放疗敏感性。本研究旨在探讨小分子EGFR酪氨酸激酶抑制剂吉非替尼能否增加肺癌细胞株HCC827和H358的放疗敏感性及其可能的机制。方法选取HCC827和H358这两个非小细胞肺癌细胞株，分为单纯X线组和X线+吉非替尼两组。单纯X线组采用单纯X线照射，X线+吉非替尼组经1 μmol/L吉非替尼作用24 h后再行X线照射。克隆形成实验比较两株细胞中不同分组细胞放射敏感性，免疫荧光激光共聚焦显微镜观察X线照射后各时间点细胞核中的磷酸化H2AX（γ-H2AX）及EGFR焦点在细胞中的定位情况，Western blot法检测放疗后胞质胞核蛋白中EGFR的表达。结果克隆形成实验中，H358细胞实验组与对照组在各放疗剂量点的SF2值分别为0.355和0.433；HCC827细胞实验组与对照组在各放疗剂量点的SF2值分别为0.223和0.242，差别不明显。激光共聚焦显微镜观察照射4 Gy后各时间段实验组H358细胞核中g-H2AX斑点相比对照组要多，且持续时间更长。而对照组和实验组的HCC827细胞g-H2AX斑点在各时间段并无明显差异；激光共聚焦显微镜观察照射4 Gy后对照组H358的EGFR蛋白在1 h内入核，而经吉非替尼处理后EGFR蛋白几乎不入核；实验组及对照组HCC827细胞的EGFR表达位置均在细胞质中，胞核中很少或者没有，可以认为并无入核现象；Western blot结果显示，H358细胞在经4 Gy放射处理后有入核现象，而预先经吉非替尼处理后，EGFR蛋白几乎不在核内表达而仍位于细胞浆内。对于HCC827细胞，实验组及对照组的EGFR蛋白均在细胞质中表达，胞核中很少或没有，且两组并无明显差异。结论吉非替尼可增加肺癌细胞株H358的放射敏感性，这可能与其阻止放疗后EGFR入核、影响放疗后双链断裂（double strand break, DSB）修复有关；而对HCC827细胞无影响，可能与其放疗后EGFR不入核相关。