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S.K. Lam
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P1.07 - Poster Session with Presenters Present (ID 459)
- Event: WCLC 2016
- Type: Poster Presenters Present
- Track: SCLC/Neuroendocrine Tumors
- Presentations: 1
- Moderators:
- Coordinates: 12/05/2016, 14:30 - 15:45, Hall B (Poster Area)
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P1.07-029 - In Vitro Effects of Pegylated Arginase in Small Cell Lung Cancer (ID 4163)
14:30 - 14:30 | Author(s): S.K. Lam
- Abstract
Background:
Small cell lung cancer (SCLC) accounts for 15% of all lung cancer cases. SCLC is notoriously difficult to treat with high relapse rate and the current standard treatment remains chemotherapy. Arginine is an important amino acid in normal human cells that can be replenished through urea cycle, but some tumors are arginine-auxotrophic due to deficient argininosuccinate synthetase (ASS1) and/or ornithine transcarbamylase (OTC). BCT-100 is a pegylated arginase, which converts arginine to citrulline, has demonstrated anticancer activity in human melanoma, hepatocellular carcinoma and acute myeloid leukemia. We aim to determine the in vitro effects of BCT-100 in SCLC.
Methods:
A panel of 7 SCLC cell lines was obtained from ATCC. Cell viability was detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and protein expression by Western blot. Knockdown of OTC was performed using siRNA. Flow cytometry was applied to detect mitochondrial membrane depolarization (MMD).
Results:
The IC~50~ values of BCT-100 in H69, DMS79, H187, H209, H526, H841 and SW1271 cells were 462.9±112.2, >1000, 24.9±6.4, 8.6±0.8, 10.1±0.7, >1000 and 49.2±7.4 mU/mL respectively. Overexpression of ASS1 in H69 and DMS79 cells, and OTC in H841 cells were associated with resistance to BCT-100. Knocking down of OTC increased sensitivity of BCT-100 in H841 cells partially via apoptosis. H526 cells (BCT-100-sensitive) was selected for mechanistic study. MMD was observed in BCT-100 treatment accompanied by cytochrome c and SMAC release from mitochondria to cytosol. N-acetylcysteine (NAC) could significantly reverse apoptosis induced by BCT-100. Besides, cyclin A2, cyclin B1 and CDK7 were downregulated in a time-dependent manner. The protein expression of p-AKT and p-mTOR was increased after exposure while RAS/RAF/ERK cell signaling pathway was inhibited with BCT-100 treatment.
Conclusion:
SCLC cell lines with low ASS1 and OTC expression were sensitive to arginine depletion with BCT-100, mediated through oxidative stress, cell cycle arrest and apoptotic pathway.
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P3.01 - Poster Session with Presenters Present (ID 469)
- Event: WCLC 2016
- Type: Poster Presenters Present
- Track: Biology/Pathology
- Presentations: 1
- Moderators:
- Coordinates: 12/07/2016, 14:30 - 15:45, Hall B (Poster Area)
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P3.01-043 - Inhibition of Ornithine Decarboxylase Facilitates Pegylated Arginase Treatment in Lung Adenocarcinoma Xenograft Models (ID 4859)
14:30 - 14:30 | Author(s): S.K. Lam
- Abstract
Background:
Arginine depletion has shown promising anticancer effects among arginine auxotrophic cancers that are deficient in argininosuccinate synthetase (ASS) and/or ornithine transcarbamylase (OTC). Pegylated arginase (PEG-BCT-100 (rhArg1peg5000)) works as an arginine depletor by converting arginine to ornithine. However, accumulated ornithine can be channeled via ornithine decarboxylase (ODC) to produce polyamines that are known to promote tumor growth. We postulate that ODC inhibition could rescue anticancer effects of BCT-100 in lung adenocarcinoma.
Methods:
A panel of 7 lung adenocarcinoma cell lines (H23, H358, HCC827, H1650, H1975, HCC2935 and HCC4006) was used to study the in vitro effect of BCT-100 by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The in vivo effect of BCT-100 was studied using 5 nude mice xenograft models lines (H358, HCC827, H1650, H1975 and HCC4006). Protein expression and arginine concentration were investigated by Western blot and ELISA respectively. TUNEL assay was performed to identify apoptosis.
Results:
BCT-100 reduced in vitro cell viability across different cell lines. However, BCT-100 could only suppress tumor growth in HCC4006 xenograft model, while paradoxical growth stimulation was observed in H358, HCC827, H1650 and H1975 xenograft models. Upon BCT-100 treatment, ODC was induced in two solid tumor xenograft models (H1650 and H1975), while unaltered in cystic tumor xenograft models (H358 and HCC827) and the remaining solid tumor (HCC4006) xenograft model. In both H1650 and H1975 xenografts, combined BCT-100 and α-Difluoromethylornithine (DFMO, an ODC inhibitor) significantly suppressed tumor growth compared with control or single arm treatments with median survival doubled compared with control group. Apoptosis was activated in combination arm in both xenograft models. In HCC4006 xenograft model, the tumor suppression effect of BCT-100 arm and DFMO/BCT-100 arm was similar. Apoptosis was noted in DFMO, BCT-100 and DFMO/BCT arms.
Conclusion:
Inhibition of ODC by DFMO is essential in BCT-100 (pegylated arginase) treatment in lung adenocarcinoma.
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P3.03 - Poster Session with Presenters Present (ID 473)
- Event: WCLC 2016
- Type: Poster Presenters Present
- Track: Mesothelioma/Thymic Malignancies/Esophageal Cancer/Other Thoracic Malignancies
- Presentations: 1
- Moderators:
- Coordinates: 12/07/2016, 14:30 - 15:45, Hall B (Poster Area)
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P3.03-018 - Suppression of Tumor Growth by Pegylated Arginase in Malignant Pleural Mesothelioma (ID 4854)
14:30 - 14:30 | Author(s): S.K. Lam
- Abstract
Background:
Malignant pleural mesothelioma (MPM) is a global health issue. Pegylated arginase (PEG-BCT-100) has shown anti-tumor effects in hepatocellular carcinoma, acute myeloid leukemia and human melanoma. We aimed to study the preclinical anticancer effects of BCT-100 in MPM.
Methods:
A panel of 5 mesothelioma cell lines (from ATCC) was used to study the in vitro effect of BCT-100 by crystal violet staining. The in vivo effects of BCT-100 (± chemotherapy) were studied using two nude mice xenograft models. Protein expression and arginine concentration were evaluated by Western Blot and ELISA respectively. Cellular location of BCT-100 was detected by immunohistochemistry and immunoflorescence staining. TUNEL assay was used to identify cellular apoptotic events.
Results:
BCT-100 reduced in vitro cell viability (IC~50~: 13-24 mU/ml) across different cell lines and suppressed tumor growth in both 211H and H226 xenograft models. Argininosuccinate synthetase was expressed in H28, H226, H2452 cells as well as 211H and H266 xenografts. Ornithine transcarbamylase was undetectable in all cell lines and xenograft models. BCT-100 (60 mg/kg) significantly suppressed tumor growth with increased median survival in both xenograft models. No beneficial effect was observed when combining BCT-100 with pemetrexed or cisplatin. BCT-100 decreased serum and intratumoral arginine level. BCT-100 was mainly located in cytosol of tumor cells. Apoptosis (PARP cleavage in 211H xenograft, Bcl-2 downregulation and cleavage of PARP and caspase 3 in H226 xenograft as well as TUNEL-positive staining in both xenografts) and G1 arrest (downregulation of cyclin A2, D3, E1 and CDK4 in 211H xenograft and suppression of cyclin A2, E1, H and CDK4 in H226 xenograft) were evident with BCT-100 treatment. Furthermore, proliferative factor Ki67 was downregulated in BCT-100 treatments arms.
Conclusion:
MPM tumor growth was suppressed by BCT-100 via apoptosis and G1 arrest in vivo. This provides scientific evidence to support further clinical exploration of BCT-100 in treatment of MPM. (Acknowledgment: This research was supported by Hong Kong Pneumoconiosis Compensation Fund Board, HKSAR.)
