Structure-activity studies of quinuclidinone analogs as anti-proliferative agents in lung cancer cell lines

We have synthesized and tested novel quinuclidinone analogs to assay the effects on H1299 lung cancer cell lines alone or with gamma-radiation. We have found two series of quinuclidinone analogs that act as anti-cancer agents. Of these, four interesting analogs significantly decreased cell viability in H1299 lung cancer cell lines. Two derivatives decreased cell proliferation in a dose-dependent fashion alone or in the presence of gamma-radiation. Radiosensitization increased when derivative treatment preceded radiation treatment for both derivatives. These preliminary studies show an evidence for both additive and synergistic cytotoxicity for treatment of lung cancer by these novel quinuclidinone analogs.     

A novel GFP nude rat model to investigate tumor-stroma interactions

The anticancer effects of ceramide have been reported in many types of cancers but less in lung cancer. In this study, we used C₂-ceramide to further investigate its possible anticancer effects and mechanisms on non-small cell lung cancer (NSCLC) H1299 cells. The result of cell proliferation in terms of trypan blue assay showed high dose of C₂-ceramide inhibited cell survival after 24 h treatment. The flow cytometry-based assays indicated the effect of apoptosis, chromatin condensation, and G₁ arrest in terms of Annexin V/propidium iodide (PI), DAPI, and PI stainings, respectively. Moreover, the decreased protein level of p-Akt, p-NFκB, survivin and cyclin A2 were detected by Western blot assay. Taken together, these results indicated the antiproliferative effect of C₂-ceramide is majorly responsible for cell apoptosis in lung cancer H1299 cells.     

Δ133p53 decreases the chemosensitivity of carcinoma cell line H1299

The research evaluated the effect of Δ133p53 on the chemosensitivity of lung adenocarcinoma cell line H1299. By this study, the drug‐resistant molecular marker and a new target for cancer therapy could be provided. Δ133p53 or negative control plasmid were transferred into H1299 cells by lentivirus vector. The expression of Δ133p53 in transfected cells was examined using immunofluorescence. The 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide (MTT) method and colony formation test were applied to detect drug sensitivity after cisplatin or 5‐fluorouracil (5‐FU) treatment. After cisplatin (CDDP)/FU treatment, MTT assay demonstrated that the inhibition rate of H1299/Δ133p53 cell was reduced compared with that of the H1299 and H1299/NEG cells at the same concentration of drug. The 50% inhibitory concentrations (IC ₅₀) of CDDP and 5‐FU rose by 36.1 and 30.2%, respectively (P < 0.05). The colony formation assay suggested that the cell proliferation ability of H1299/Δ133p53 cell was prominently increased when compared with that of control group H1299 and H1299 /NEG cells (P < 0.05). The present study demonstrated that the transfection of the Δ133p53 gene in H1299 cells led to the reduction of chemosensitivity.     

Effects of Cinobufagin on the Proliferation,Migration, and Invasion of H1299 Lung Cancer Cells

Cinobufagin (CB), with its steroidal nucleus structure, is one of the major, biologically active components of Chan Su. Recent studies have shown that CB exerts inhibitory effects against numerous cancer cells. However, the effects of CB regarding the metastasis of non-small cell lung cancer (NSCLC) and the involved mechanisms need to be further studied. The purpose of the present study aimed to report the inhibitory function of CB against proliferation and metastasis of H1299 cells. CB inhibited proliferation of H1299 lung cancer cells with an IC₅₀ value of 0.035±0.008 μM according to the results of MTT assays. Antiproliferative activity was also observed in colony forming cell assays. In addition, 5-ethynyl-2’-deoxyuridine (EdU) retention assays revealed that CB significantly inhibited the rate of DNA synthesis in H1299 cells. Moreover, results of the scratch wound healing assays and transwell migration assays displayed that CB exhibited significant inhibition against migration and invasion of H1299 cells. Furthermore, CB could concentration-dependently reduce the expression of integrin α2, β-catenin, FAK, Src, c-Myc, and STAT3 in H1299 cells. These western blotting results indicated that CB might target integrin α2, β-catenin, FAK and Src to suppress invasion and migration of NSCLC, which was consistent with the network pharmacology analysis results. Collectively, findings of the current study suggest that CB possesses promising activity against NSCLC growth and metastasis.     

Synthesis and Cytotoxicity Evaluation of Panaxadiol Derivatives

In this study, 13 panaxadiol (PD) derivatives were synthesized via reactions with aromatic compounds and amino acids. Following this, the cytotoxicity of these compounds was evaluated against four cancer cell lines (human hepatoma cells HepG‐2, human lung cancer cells A549, human breast cancer cells MCF‐7, and human colon cancer cells HCT‐116) and one normal cell lines (human gastric epithelial cells GES‐1). The results showed that the panaxadiol derivatives 3 , 12 , and 13 showed significant inhibition of cellular proliferation against cancer cells compared with PD, and the panaxadiol derivative 12 had the lowest IC₅₀ value for A549 (IC₅₀=18.91±1.03 μm ). For MCF‐7 cells, most compounds exhibited good inhibition of cellular proliferation, and the panaxadiol derivative 13 showed the strongest inhibitory effect (IC₅₀=8.62±0.23 μm ), which significantly increased the cytotoxicity of PD and was stronger than the positive control (mitomycin). For normal cells, all compounds exhibited low or no toxic effects; thus, these derivatives can be used to develop novel antiproliferative agents.     

A novel cytostatic form of autophagy in sensitization of non-small cell lung cancer cells to radiation by vitamin D and the vitamin D analog,EB 1089

The standard of care for unresectable lung cancer is chemoradiation. However, therapeutic options are limited and patients are rarely cured. We have previously shown that vitamin D and vitamin D analogs such as EB 1089 can enhance the response to radiation in breast cancer through the promotion of a cytotoxic form of autophagy. In A549 and H460 non-small cell lung cancer (NSCLC) cells, 1,25-D₃ (the hormonally active form of vitamin D) and EB 1089 prolonged the growth arrest induced by radiation alone and suppressed proliferative recovery, which translated to a significant reduction in clonogenic survival. In H838 or H358 NSCLC cells, which lack VDR/vitamin D receptor or functional TP53, respectively, 1,25-D₃ failed to modify the extent of radiation-induced growth arrest or suppress proliferative recovery post-irradiation. Sensitization to radiation in H1299 NSCLC cells was evident only when TP53 was induced in otherwise tp53-null H1299 NSCLC cells. Sensitization was not associated with increased DNA damage, decreased DNA repair or an increase in apoptosis, necrosis, or senescence. Instead sensitization appeared to be a consequence of the conversion of the cytoprotective autophagy induced by radiation alone to a novel cytostatic form of autophagy by the combination of 1,25-D₃ or EB 1089 with radiation. While both pharmacological and genetic suppression of autophagy or inhibition of AMPK phosphorylation sensitized the NSCLC cells to radiation alone, inhibition of the cytostatic autophagy induced by the combination treatment reversed sensitization. Evidence for selectivity was provided by lack of radiosensitization in normal human bronchial cells and cardiomyocytes. Taken together, these studies have identified a unique cytostatic function of autophagy that appears to be mediated by VDR, TP53, and possibly AMPK in the promotion of an enhanced response to radiation by 1,25-D₃ and EB 1089 in NSCLC.     

Isolation of multiple cell-binding ligands from different phage displayed-peptide libraries

A technical challenge in the development of biosensor devices for cancer detection and diagnosis is the identification of ligands that recognize cancer cells with high affinity and specificity. Furthermore, it is unlikely that one cell-binding ligand will provide sufficient biological information, thus, multiple ligands for a given cancer type will be needed for confident clinical diagnosis. Biopanning of phage displayed peptide libraries is a route to isolation of specific cell-binding reagents. A potential approach towards isolation of multiple ligands for a single cell type is to pan against the same cell type using different peptide libraries. Here we report the synthesis of a new 20-mer peptide-phage library and its use to select a peptide that binds to the large cell lung carcinoma cell line, H1299. The isolated phage clone binds H1299 cells 80 times better than a control phage and can distinguish between H1299 and normal control cells. The phage clone also binds to the lung pleura epidermoid cell line, Calu-1 but not to all lung carcinoma cell lines. The peptide is functional outside the context of the phage and tetramerization of the peptide on a trilysine core improves the affinity of the peptide. The tetrameric peptide can be used to deliver a fluorescent quantum dot to H1299 cells. Unexpectedly, the peptide shares sequence similarity to a previously isolated H1299-binding peptide isolated from a different 20-mer peptide library. Data suggests that the two peptides target the same cellular receptor. Our results imply that cell-based biopanning can isolate cell-binding ligands that may be of utility for cancer diagnosis, and isolation of cell-targeting peptides from different peptide libraries can expand the repertoire of cell-binding reagents.     

Cx31.1 acts as a tumour suppressor in non-small cell lung cancer (NSCLC) cell lines through inhibition of cell proliferation and metastasis

Reduced connexin expression and loss of gap junction function is a characteristic of many cancers, including lung cancer. However, there are little reports about the relation between Cx31.1 and lung cancer. This study was conducted to investigate the effect of Cx31.1 on non-small cell lung cancer (NSCLC). We found that the Cx31.1 was down-regulated in NSCLC cell lines, and the expression levels were reversely related with their metastatic potential. We ectopically expressed Cx31.1 in H1299 NSCLC cell line to examine the influence of Cx31.1 overexpression. The results showed that overexpression of Cx31.1 in H1299 cells reduced cell proliferation, induced a delay in the G(1) phase, inhibited anchorage-independent growth and suppressed cell migration and invasion. The cell cycle delay and cell migration and invasion suppressive effects of Cx31.1 were partially reversed by siRNA targeting mRNA of Cx31.1. Moreover, xenografts of Cx31.1 overexpressing H1299 cells showed reduced tumourigenicity. These results suggested that Cx31.1 has tumour-suppressive properties. Further investigation indicated that cyclin D3 may be responsible for Cx31.1-induced G(1) phase delay. Importantly, Cx31.1 increased the expression of epithelial markers, such as cytokeratin 18, and decreased expression of mesenchymal markers, such as vimentin, indicating a Cx31.1-mediated partial shift from a mesenchymal towards an epithelial phenotype. We concluded that Cx31.1 inhibit the malignant properties of NSCLC cell lines, the mechanisms under this may include regulation of EMT.     

Transient Transfection of a Wild-Type p53 Gene Triggers Resveratrol-Induced Apoptosis in Cancer Cells

Resveratrol is a promising chemopreventive agent that mediates many cellular targets involved in cancer signaling pathways. p53 has been suggested to play a role in the anticancer properties of resveratrol. We investigated resveratrol-induced cytotoxicity in H1299 cells, which are non-small lung cancer cells that have a partial deletion of the gene that encodes the p53 protein. The results for H1299 cells were compared with those for three cell lines that constitutively express wild-type p53: breast cancer MCF-7, adenocarcinomic alveolar basal epithelia A549 and non-small lung cancer H460. Cell viability assays revealed that resveratrol reduced the viability of all four of these cell lines in a dose- and time-dependent manner. MCF-7, A549 and H460 cells were more sensitive to resveratrol than were H1299 cells when exposed to the drug for 24 h at concentrations above 100 µM. Resveratrol also increased the p53 protein levels in MCF-7 cells without altering the p53 mRNA levels, suggesting a post-translational modulation of the protein. The resveratrol-induced cytotoxicity in these cells was partially mediated by p53 and involved the activation of caspases 9 and 7 and the cleavage of PARP. In H1299 cells, resveratrol-induced cytotoxicity was less pronounced and (in contrast to MCF-7 cells) cell death was not accompanied by caspase activation. These findings are consistent with the observation that MCF-7 cells were positively labeled by TUNEL following exposure to 100 µM resveratrol whereas H1299 cells under similar conditions were not labeled by TUNEL. The transient transfection of a wild-type p53-GFP gene caused H1299 cells to become more responsive to the pro-apoptotic properties of resveratrol, similarly to findings in the p53-positive MCF-7 cells. Our results suggest a possible therapeutic strategy based on the use of resveratrol for the treatment of tumors that are typically unresponsive to conventional therapies because of the loss of normal p53 function.     

A Comparison of the Biological Effects of 125I Seeds Continuous Low-Dose-Rate Radiation and 60Co High-Dose-Rate Gamma Radiation on Non-Small Cell Lung Cancer Cells

Objectives

To compare the biological effects of ¹²⁵I seeds continuous low-dose-rate (CLDR) radiation and ⁶⁰Co γ-ray high-dose-rate (HDR) radiation on non-small cell lung cancer (NSCLC) cells.

Materials and Methods

A549, H1299 and BEAS-2B cells were exposed to ¹²⁵I seeds CLDR radiation or ⁶⁰Co γ-ray HDR radiation. The survival fraction was determined using a colony-forming assay. The cell cycle progression and apoptosis were detected by flow cytometry (FCM). The expression of the apoptosis-related proteins caspase-3, cleaved-caspase-3, PARP, cleaved-PARP, BAX and Bcl-2 were detected by western blot assay.

Results

After irradiation with ¹²⁵I seeds CLDR radiation, there was a lower survival fraction, more pronounced cell cycle arrest (G₁ arrest and G₂/M arrest in A549 and H1299 cells, respectively) and a higher apoptotic ratio for A549 and H1299 cells than after ⁶⁰Co γ-ray HDR radiation. Moreover, western blot assays revealed that ¹²⁵I seeds CLDR radiation remarkably up-regulated the expression of Bax, cleaved-caspase-3 and cleaved-PARP proteins and down-regulated the expression of Bcl-2 proteins in A549 and H1299 cells compared with ⁶⁰Co γ-ray HDR radiation. However, there was little change in the apoptotic ratio and expression of apoptosis-related proteins in normal BEAS-2B cells receiving the same treatment.

Conclusions

¹²⁵I seeds CLDR radiation led to remarkable growth inhibition of A549 and H1299 cells compared with ⁶⁰Co HDR γ-ray radiation; A549 cells were the most sensitive to radiation, followed by H1299 cells. In contrast, normal BEAS-2B cells were relatively radio-resistant. The imbalance of the Bcl-2/Bax ratio and the activation of caspase-3 and PARP proteins might play a key role in the anti-proliferative effects induced by ¹²⁵I seeds CLDR radiation, although other possibilities have not been excluded and will be investigated in future studies.     

A novel cytostatic form of autophagy in sensitization of non-small cell lung cancer cells to radiation by vitamin D and the vitamin D analog,EB 1089

The standard of care for unresectable lung cancer is chemoradiation. However, therapeutic options are limited and patients are rarely cured. We have previously shown that vitamin D and vitamin D analogs such as EB 1089 can enhance the response to radiation in breast cancer through the promotion of a cytotoxic form of autophagy. In A549 and H460 non-small cell lung cancer (NSCLC) cells, 1,25-D₃ (the hormonally active form of vitamin D) and EB 1089 prolonged the growth arrest induced by radiation alone and suppressed proliferative recovery, which translated to a significant reduction in clonogenic survival. In H838 or H358 NSCLC cells, which lack VDR/vitamin D receptor or functional TP53, respectively, 1,25-D₃ failed to modify the extent of radiation-induced growth arrest or suppress proliferative recovery post-irradiation. Sensitization to radiation in H1299 NSCLC cells was evident only when TP53 was induced in otherwise tp53-null H1299 NSCLC cells. Sensitization was not associated with increased DNA damage, decreased DNA repair or an increase in apoptosis, necrosis, or senescence. Instead sensitization appeared to be a consequence of the conversion of the cytoprotective autophagy induced by radiation alone to a novel cytostatic form of autophagy by the combination of 1,25-D₃ or EB 1089 with radiation. While both pharmacological and genetic suppression of autophagy or inhibition of AMPK phosphorylation sensitized the NSCLC cells to radiation alone, inhibition of the cytostatic autophagy induced by the combination treatment reversed sensitization. Evidence for selectivity was provided by lack of radiosensitization in normal human bronchial cells and cardiomyocytes. Taken together, these studies have identified a unique cytostatic function of autophagy that appears to be mediated by VDR, TP53, and possibly AMPK in the promotion of an enhanced response to radiation by 1,25-D₃ and EB 1089 in NSCLC.     

Effect of protein-tyrosine phosphatase 4A3 by small interfering RNA on the proliferation of lung cancer

Previous studies on PTP4A3 mainly focused on tumor metastasis due to the close relationship between the overexpression of lung cancer and metastasis. However, the role of PTP4A3 in the proliferation of tumor still has remained unclear. To investigate the role of PTP4A3 in cell growth of lung cancer, we constructed PTP4A3-siRNA expressing lentivirus and infected human lung cancer H1299 cells, and then examined the inhibitory effect of PTP4A3 in vitro. The levels of PTP4A3 mRNA and protein in H1299 cells decreased after PTP4A3-siRNA lentivirus infection. The growth and colony formation of the infected cells were also inhibited, indicating that PTP4A3 gene is closely associated with the proliferation of H1299 cells. In addition, after PTP4A3 specific siRNA lentivirus infection, it was notable that whilst H1299 cells in G1 phase apparently reduced, both of H1299 cells in G2/M phase and the cell apoptosis increased significantly. This finding indicated the close relationship between PTP4A3 gene and apoptosis in the H1299 cells. These results come to their conclusion that PTP4A3 plays an important role in the growth of lung cancer cells. PTP4A3 may be considered as a valuable target for anti-tumor therapeutic strategies.     

Carbamate derivatives of colchicine show potent activity towards primary acute lymphoblastic leukemia and primary breast cancer cells—in vitro and ex vivo study

Colchicine ( COL ) shows strong anticancer activity but due to its toxicity towards normal cells its wider application is limited. To address this issue, a library of 17 novel COL derivatives, namely N‐carbamates of N‐deacetyl‐4‐(bromo/chloro/iodo)thiocolchicine, has been tested against two types of primary cancer cells. These included acute lymphoblastic leukemia (ALL) and human breast cancer (BC) derived from two different tumor subtypes, ER+ invasive ductal carcinoma grade III (IDCG3) and metastatic carcinoma (MC). Four novel COL derivatives showed higher anti‐proliferative activity than COL (IC₅₀ = 8.6 nM) towards primary ALL cells in cell viability assays (IC₅₀ range of 1.1‐6.4 nM), and several were more potent towards primary IDCG3 (IC₅₀ range of 0.1 to 10.3 nM) or MC (IC₅₀ range of 2.3‐9.1 nM) compared to COL (IC₅₀ of 11.1 and 11.7 nM, respectively). In addition, several derivatives were selectively active toward primary breast cancer cells compared to normal breast epithelial cells. The most promising derivatives were subsequently tested against the NCI panel of 60 human cancer cell lines and seven derivatives were more potent than COL against leukemia, non–small‐cell lung, colon, CNS and prostate cancers. Finally, COL and two of the most active derivatives were shown to be effective in killing BC cells when tested ex vivo using fresh human breast tumor explants. The present findings indicate that the select COL derivatives constitute promising lead compounds targeting specific types of cancer.     

Ligularia fischeri regulates lung cancer cell proliferation and migration through down-regulation of epidermal growth factor receptor and integrin β1 expression

In the present study, we report the effect and molecular mechanism of Ligularia fischeri (LF) on proliferation and migration in human lung cancer cells. LF-mediated inhibition of cell proliferation in p53 wild-type A549 and p53-deficient H1299 cells is accompanied by reduced expression of cell cycle-related proteins such as cyclin-dependent kinases and cyclins, resulting in pRb hypophosphorylation and G₁ phase cell cycle arrest. In contrast, LF inhibits cell migration in A549 cells, but not in H1299 cells. These regulatory effects of LF on cell proliferation and migration are associated with inactivation of mitogenic signaling pathways such as ERK, Akt and p70^S6K, and down-regulation of epidermal growth factor receptor and integrin β1 expression. Collectively, these findings suggest further development and evaluation of LF for the prevention and treatment of lung cancer with mutated p53 as well as wild-type p53.     

Caffeine induces cytoskeletal changes and cell death in H1299 cells

Caffeine is the most common natural neuroactive substance around the world. The exact mechanism of the anticancer effects of caffeine is not clear, especially in the contexts of the cytoskeletal changes. It is known that caffeine exerts an effect on cell cycle, cell proliferation, radiosensivity of cells, and also induces cell death. The aim of the study was to determine the effect of 10 and 20 mM L^?1 caffeine on the major cytoskeletal proteins in non-small lung cancer cell line H1299. Caffeine treatment induced abnormalities in morphology and ultrastructure of cells. Moreover, the fluorescence studies showed changes in organization of vimentin, β-tubulin, lamin A/C and F-actin, which were attributed to the induction of cell death. The results also demonstrated that caffeine induced formation of two cell populations: giant, mono- or multinucleated cells, with the phenotype of mitotic catastrophe and shrunken cells with condensation of chromatin, typical of apoptosis. This study for the first time shows the effect of caffeine on the cytoskeleton of H1299 cell line. In conclusion, a high-dose caffeine treatment induces apoptotic cell death and makes it a powerful anticancer agent that should be considered for the treatment of non-small cell lung cancer.     

Ganoderic acid Me induces G1 arrest in wild-type p53 human tumor cells while G1/S transition arrest in p53-null cells

The mechanism of cell cycle arrest of tumor cells induced by ganoderic acid Me (GA-Me) is not understood. In this work, GA-Me was found to possess remarkable cytotoxicity on highly metastatic lung carcinoma 95-D cell line in both dose- and time-dependent manners. The effect of GA-Me on cell cycle arrest was found in 95-D, p53-null lung cancer cells H1299, HCT-116 p53^+/+ and HCT-116 p53^?/? human colon cancer cells. To obtain an insight into the role of p53 in cell cycle arrest by GA-Me, 95-D, H1299, HCT-116 p53^+/+ and HCT-116 p53^?/? cells were used for further investigation. GA-Me arrested cell cycle at G₁ phase in 95-D and HCT-116 p53^+/+ cells while S phase or G₁/S transition arrest in H1299 and HCT-116 p53^?/? cells. The results suggested that p53 may be a target of GA-Me, and it may be looked at as a new promising candidate for the treatment of carcinoma cells.     

Inhibition of RAC1-GEF DOCK3 by miR-512-3p contributes to suppression of metastasis in non-small cell lung cancer

MicroRNAs are a class of small non-coding RNAs regulating gene expression. In this study, we demonstrated that retinoic acid (RA) treatment increases the expression of miR-512-3p. Overexpression of miR-512-3p inhibited cell adhesion, migration, and invasion in non-small cell lung cancer (NSCLC) cell lines A549 and H1299. miR-512-3p inhibitor partially reversed these effects in H1299 cells stably expressing miR-512. We identified DOCK3, a RAC1-GEF (guanine nucleotide exchange factor), as a target gene of miR-512-3p. Overexpression of miR-512-3p led to the decrease of DOCK3 protein but not its mRNA. Knockdown of DOCK3 resulted in similar effects on adhesion, migration, and invasion as observed of miR-512-3p overexpression. Active RAC1 pull-down assay indicated that overexpression of miR-512-3p could decrease the activity of RAC1 with a higher efficiency than that of DOCK3 knockdown. Furthermore, expression of miR-512-3p was suppressed in most NSCLC patient tumor samples compared to its paired normal controls, suggesting that miR-512-3p might play a crucial role in lung cancer development. In conclusion, our results supported that miR-512-3p could inhibit tumor cell adhesion, migration, and invasion by regulating the RAC1 activity via DOCK3 in NSCLC A549 and H1299 cell lines.     

A novel carbazole derivative, BMVC: a potential antitumor agent and fluorescence marker of cancer cells

We have investigated a novel compound, 3,6-bis[2-(1-methylpyridinium)vinyl]carbazole diiodide (BMVC), for inhibiting telomerase activity and distinguishing human lung H1299 and oral Ca9-22 cancer cells from lung IMR90 and skin Detroit-551 normal fibroblast cells. The telomeric repeat amplification protocol (TRAP) assay shows that the concentration of BMVC that inhibits 50% of the telomerase activity (IC50) is ca. 0.05 microM. On the other hand, the cell-viability assay indicates that the cytotoxicity was less than 15% to the H1299 and Ca9-22 cancer cells, and almost negligible to the MRC-5 and Detroit-551 normal cells after incubation with 0.5 microM BMVC for 72 h. The low concentration of 0.05 microM of BMVC can inhibit telomerase activity but does not have general toxic effects to normal cells, implying that BMVC is a promising telomerase inhibitor. Moreover, wide-field fluorescence images of 0.1 microM BMVC-treated cells show bright fluorescence spots in the nuclei of the most H1299 and Ca9-22 cancer cells. Interestingly, similar fluorescence spots are hardly observed in the nuclei of the IMR90 and Detroit-551 normal cells, implying that BMVC might be a useful marker to distinguish tumor cells and normal cells.