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S.G. Gray
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MINI 08 - Prognostic/Predictive Biomarkers (ID 106)
- Event: WCLC 2015
- Type: Mini Oral
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
- Moderators:T.E. Stinchcombe, N. Pavlakis
- Coordinates: 9/07/2015, 16:45 - 18:15, Mile High Ballroom 4a-4f
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MINI08.08 - VEGF-Mediated Cell Survival in NSCLC: Implications for Epigenetic Targeting of VEGF Receptors as a Therapeutic Approach (ID 2721)
16:45 - 18:15 | Author(s): S.G. Gray
- Abstract
- Presentation
Background:
We have recently shown that VEGF, at least in part, is an autocrine growth factor for NSCLC cells, mediating its survival effects via VEGFR2 (KDR) in addition to the more novel receptor, Neuropilin-1 (Barr et al., Mol Cancer, 2015). In this study, we evaluated the potential therapeutic utility of histone deacetylase (HDAC) inhibitors in targeting the VEGF-VEGFR signalling axis in non-small cell lung cancer (NSCLC) cells.
Methods:
The effect of the HDAC inhibitor, Trichostatin-A (TSA) on modulating the expression of the VEGF receptors, VEGFR1, VEGFR2, NP1 and NP2, in A549 and SKMES-1 cells was examined and validated at the mRNA level and protein levels using RT-PCR and Western blot analysis. Gene expression was further validated by quantitative real-time PCR. To investigate the effect of TSA on the viability of NSCLC cells, these were treated with increasing concentrations of TSA (2.5 ng/ml-250 ng/ml) for 24h. Cell proliferation and apoptosis was measured by BrdU and Annexin V/PI (FACS), respectively. VEGF protein secretion in response to TSA was assessed in conditioned media from lung tumour cells by ELISA. To determine if the effects of TSA on VEGFR receptors were mediated through immediate to early responses, cells were pre-treated with cycloheximide (10 µg/ml) for 2 h followed by treatment with TSA (250 ng/ml) for 24 h. To confirm whether the observed effects of HDAC inhibition by TSA were due to increased histone hyperacetylation at the VEGFR1 and VEGFR2 gene promoters, chromatin immunoprecipitation (ChIP) analysis was carried out following treatment with TSA.
Results:
NP1 and NP2 mRNA levels were decreased in both A549 and SKMES-1 lung cancer cells in response to TSA and induced the expression of VEGFR1 and VEGFR2 at higher concentrations. TSA however, had no effect on VEGF mRNA expression. Critically, the effect of TSA was more marked at the protein level, with complete loss of Neuropilin-1 protein. HDAC inhibition resulted in a significant decrease in the viability of A549 and SKMES-1 cells in a dose-dependent fashion. While TSA induced significant apoptosis of both lung tumour cell lines, VEGF was unable to rescue cells from TSA-induced cell death. VEGF secretion was significantly decreased in both cell lines. Treatment with cycloheximide was unable to abrogate the TSA-mediated increase in the VEGF receptors examined, indicating that de novo protein synthesis is not required for these observed effects, but may be due to direct effects at the promoter level. Direct histone acetylation of histones H3 and H4 was observed, indicating an increase in histone hyperacetylation of VEGFR1 and VEGR2 promoters. A significant trend in the modulation of the VEGF receptors similar to that seen in response to TSA was shown when treated with Vorinostat (SAHA).
Conclusion:
Epigenetic targeting of the Neuropilin receptors may offer an effective treatment for NSCLC patients in the clinical setting. The possibility of novel targeted agents decreasing the levels, or function, of tumour VEGF receptors, in particular NP1, may lead to more successful treatments and prolonged overall survival in these patients.
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P1.08 - Poster Session/ Thymoma, Mesothelioma and Other Thoracic Malignancies (ID 224)
- Event: WCLC 2015
- Type: Poster
- Track: Thymoma, Mesothelioma and Other Thoracic Malignancies
- Presentations: 1
- Moderators:
- Coordinates: 9/07/2015, 09:30 - 17:00, Exhibit Hall (Hall B+C)
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P1.08-031 - Induction of Protein Citrullination and Auto-Antibodies Production in Murine Exposed to Nickel Nanomaterials (ID 2827)
09:30 - 17:00 | Author(s): S.G. Gray
- Abstract
Background:
Citrullination, or the post-translational deimination of polypeptide-bound arginine, is involved in several pathological processes in the body, including autoimmunity and tumorigenesis. This enzymatic conversion is governed by the family of Ca[2+]-dependent peptidylarginine deiminases (PAD). Citrullinated proteins are recognised as non-self-proteins, and subsequently can induce an autoimmune response. Recent studies have shown that nanomaterials of diverse origin can trigger protein citrullination, which might constitute a common pathogenic link to disease development.
Methods:
Engineered nickel nanomaterials, which can mimic environmental filamentous materials were hypothesised to trigger similar pathophysiological responses. Mice were injected intraperitoneally with either nickel nanomaterials or phosphate buffered saline. Murine sera samples for anti-CCP3 detection and tissue samples for immunohistochemical analysis were collected at day 1 and day 14.
Results:
Auto-antibody production was detected in serum of nickel nanomaterials-treated mice. Citrullination-associated phenomena and PAD levels were found to be elevated in nanomaterials -treated cell lines as well as in the spleen, kidneys and lymph nodes of mice, suggesting a systemic response to nickel nanomaterials injection, and validated in human pleural and pericardial malignant mesothelioma (MM) samples.
Conclusion:
The observed systemic responses in mice exposed to nickel nanomaterials support the evidence linking exposure to environmental factors with the development of autoimmunity responses and reinforces the need for comprehensive safety screening of nanomaterials. Furthermore, these nanomaterials induce pathological processes that mimic those observed in Pleural MM, and therefore require further investigations into their carcinogenicity.
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P2.08 - Poster Session/ Thymoma, Mesothelioma and Other Thoracic Malignancies (ID 225)
- Event: WCLC 2015
- Type: Poster
- Track: Thymoma, Mesothelioma and Other Thoracic Malignancies
- Presentations: 1
- Moderators:
- Coordinates: 9/08/2015, 09:30 - 17:00, Exhibit Hall (Hall B+C)
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P2.08-014 - Therapeutic Effectiveness of MET/RON Small Molecule Inhibitor BMS-777607 on Cell Viability of Human Malignant Pleural Mesothelioma Cell Lines (ID 2832)
09:30 - 17:00 | Author(s): S.G. Gray
- Abstract
Background:
Overexpression of c-Met receptor tyrosine kinase has been highly associated with oncogenic progression of non-neoplastic mesothelial progenitor cells to malignant pleural mesothelioma (MPM). Moreover, activated c-Met receptor tyrosine kinase transduces signals that regulate tumorigenic activities including cell growth, migration, survival, and invasion of extracellular matrixes. A small molecule MET kinase inhibitor (BMS-777607) is an inhibitor of tyrosine receptor currently under clinical trials. Previous studies reported the effect of this inhibitor on cancer cells such as breast, hepatic and prostate cancer. However, its inhibitory effect on malignant pleural mesothelioma (MPM) cells has not yet been evaluated. In our study, we aimed to investigate the therapeutic usefulness of this small molecule on MPM.
Methods:
Human MPM cell lines such as REN and NCl-H2373 and a human lung carcinoma cell line (NCl-H226) were used. LP-9 cell line, which resembles normal human mesothelial cells, was used as control. These cells were cultured and exposed to BMS-777607 at concentrations 1uM, 5uM and 10uM. Cell cycle, cell viability, lysosomal mass/pH changes and mitochondrial membrane potential were examined using immunofluorescent staining methods and data were collected and analysed with high content screening systems such as Cytell and IN Cell Investigator software.
Results:
Both MPM cell lines and the lung cell line showed cell cycle arrest as examined by Cytell in significant dose-dependent manner with maximum effects seen at the highest dose (10 µM) of BMS-777607. Cell viability and other biological cellular markers were also altered upon exposure to this small molecule. Our results showed that BMS-777607 induces negligible changes of these markers examined in the normal mesothelial cells line (LP-9).
Conclusion:
Taken together, these findings indicate that inhibition of MET/RON signalling using a small molecule inhibitor such as BMS-777607 could significantly interrupt the cell cycle stages and alter other cellular compartments (i.e; lysosomal mass/pH, mitochondrial membrane potential) which lead to suppression of MPM cell viability, suggesting that such a targeting strategy may hold promise for the treatment of MPM.
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P3.08 - Poster Session/ Thymoma, Mesothelioma and Other Thoracic Malignancies (ID 226)
- Event: WCLC 2015
- Type: Poster
- Track: Thymoma, Mesothelioma and Other Thoracic Malignancies
- Presentations: 1
- Moderators:
- Coordinates: 9/09/2015, 09:30 - 17:00, Exhibit Hall (Hall B+C)
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P3.08-002 - Nanodiamonds-Conjugated Chemotherapeutic Agent Induces Cell Cycle Arrest and Apoptosis of Human Malignant Pleural Mesothelioma Cell Lines (ID 2809)
09:30 - 17:00 | Author(s): S.G. Gray
- Abstract
Background:
The use of nanomaterials-based therapeutic systems is rapidly growing and covering a several biomedical applications such as detection, diagnosis and treatment. Recently, nanodiamonds (NDs) have been demonstrated to have great potential as a multimodal imaging/therapy platform. NDs enhance the ability of the drug to cross the cell membrane, increase intracellular drug delivery to the cancer cells, improve treatment efficacy, and decrease toxicity to normal cells or tissues. NDs are attractive for use in drug delivery because of their rich surface chemistry, advantageous size, and ability to act as transmembrane carriers. NDs have also been shown to enhance therapeutic efficacy of doxorubicin (DOX), particularly in treating murine liver and mammary tumor models, and lung metastasis of breast cancer. Yet, nothing similar has been done to translate ND-DOX systems to other cancers such as malignant pleural mesothelioma (MPM). In our study, we aimed to investigate the efficacy of ND-conjugated DOX on MPM cell lines.
Methods:
Bare, uncoated nanodiamonds were pegylated and functionalised with DOX. NDs/Dox were washed three times then re-suspended in water. Nanoparticle Tracking Analysis (NTA), FTIR and PL spectroscopy and TEM imaging were performed to characterise the functionalisation. Human MPM cell lines such as REN and NCl-H2373 and human lung carcinoma cell line (NCl-H226) were used. LP-9 cell line, which resembles normal human mesothelial cells, was used as control. All cells were cultured and exposed to NDs, NDs/DOX or DOX alone. Cell cycle, cell viability, lysosomal mass/pH changes and mitochondrial membrane potential were examined using immunofluorescent staining techniques and data were collected and analysed with high content screening tools such as Cytell and IN Cell Investigator software.
Results:
Significant alterations of the examined biological markers were detected in human MPM cell lines such (REN and NCl-H2373) and human lung carcinoma cells (H226), while a slight changes were seen in the LP-9 cells. Interestingly, the MPM cells showed greater responses to NDs/DOX versus DOX alone.
Conclusion:
Our study demonstrates that NDs loaded with DOX exert significant inhibitory activities of human MPM cell lines and at concentrations that have minimal effects on normal pleura. Thus, ND-conjugated chemotherapy represents a promising, biocompatible strategy for enhancing chemotherapy efficacy and safety.
