NCI-H460 Cell Research Articles

Design, synthesis and pharmacological evaluation of new 3-(1H-benzimidazol-2-yl)quinolin-2(1H)-one derivatives as potential antitumor agents

A series of new 3-(1H-benzimidazol-2-yl)quinolin-2(1H)-one derivatives (5a1−5d6) were designed and synthesized as antitumor agents. In vitro antitumor assay results showed that some compounds exhibited moderate to high inhibitory activity against HepG2, SK-OV-3, NCI-H460 and BEL-7404 tumor cell lines, and most compounds exhibited much lower cytotoxicity against the HL-7702 normal cell line compared to 5-FU and cisplatin. In vivo antitumor assay results demonstrated that 5a3 exhibited effective inhibition on tumor growth in the NCI-H460 xenograft mouse model and that 5d3 displayed excellent antiproliferative activity in the BEL-7402 xenograft model. These results suggested that both 5a3 and 5d3 could be used as anticancer drug candidates. Mechanistic studies suggested that compounds 5a3 and 5d3 exerted their antitumor activity by up-regulation of Bax, intracellular Ca2+ release, ROS generation, downregulation of Bcl-2, activation of caspase-9 and caspase-3 and subsequent cleavage of PARP, inhibition of CDK activity and activation of the p53 protein.

Long non-coding RNA-CCAT2 promotes the occurrence of non-small cell lung cancer by regulating the Wnt/β-catenin signaling pathway.

The present study aimed to investigate the biological function of colon cancer-associated transcript 2 (CCAT2) in the occurrence and progression of non-small cell lung carcinoma (NSCLC) and its potential use in the early diagnosis and molecular-targeted therapy of NSCLC. The tumor tissues, para-carcinoma tissues and associated clinical data of 36 patients with NSCLC were collected in order to detect the expression of CCAT2 and assess the impact of factors including histopathological type, Tumor-Node-Metastasis stage and lymph node metastasis on CCAT2 expression. The lung cancer NCI-H1975 cell line was transfected with a small interfering RNA (siRNA) plasmid to determine the effect of si-CCAT2 on NSCLC proliferation, invasion and metastasis. The effect of si-CCAT2 on the expression of nuclear and cytoplasmic β-catenin protein in the lung cancer NCI-H1975 cell line was detected using western blot analysis. The expression levels of CCAT2 in the tumor tissues of patients with NSCLC were significantly higher than those in the normal para-carcinoma tissues (t=8.580, P<0.01). Subsequent to CCAT2 silencing, the proliferation and invasive abilities of NCI-H1975 cells were significantly decreased compared with control cells (P<0.05). In the si-CCAT2 group, the level of nuclear and cytoplasmic β-catenin proteins was decreased, and the activity of the Wnt signaling pathway was significantly inhibited compared with the control cells (P<0.01), and a synergistic effect was exerted with the Wnt signaling inhibitor FH535. CCAT2 may therefore promote the occurrence of NSCLC by regulating the Wnt/β-catenin signaling pathway.

Carboline alkaloids and isocoumarins from the wasp pathogenic fungus Ophiocordyceps sphecocephala BCC 2661

Ten β-carboline alkaloids, including five new and two new naturally occurring compounds, sphecolines A–G (1–7), together wit2h four known isocoumarins were isolated from a wasp-pathogenic fungus Ophiocordyceps sphecocephala BCC 2661. Structures of these compounds were determined by NMR spectroscopic and MS spectrometric analyses. Sphecolines A (1) and C (3) exhibited cytotoxic activity against human small-cell lung cancer NCI-H187 cell lines, with IC50 values of 79.9 and 75.1 μM, respectively.

Synthesis and anticancer activity of new dihydropyrimidinone derivatives

A series of dihydropyrimidinone derivatives bearing various N-heterocyclic moieties was designed and synthesized. Twelve new compounds were screened for their cytotoxic activity using 60 cancer cell lines according to NCI (USA) protocol. Compound 19 showed a significant activity against NCI-H460, SK-MEL-5, and HL-60 (TB) cell lines with growth inhibition 88%, 86% and 85%, respectively, and was found to be more safe on normal cells when compared to doxorubicin. Enzyme inhibition assay was performed for compound 19 against mTOR (IC50 = 0.64 μM) and VEGFR-2 (IC50 = 1.97 μM) to show high potency in comparison to rapamycin (IC50 = 0.43 μM) and sorafenib (IC50 = 0.3 μM) as references, respectively. Cell cycle analysis of A549 cells treated with 19 showed cell cycle arrest at G2/M phase and pro-apoptotic activity as indicated by annexin V-FITC staining.

Abstract 4114: Development and application of 18F-labeled Tenascin-C aptamer for cancer imaging

Abstract Aptamers are synthetic single-stranded DNA or RNA that can be held together with complementary sequences by base-pairing. Tenascin-C is an extracellular matrix protein that is overexpressed in cancer tissues. Herein, we investigate the biological characteristics of Tenascin-C aptamer as potential tumor imaging agents. Cy5 or F-18 labeled Tenascin-C aptamers were prepared through hybridization-based labeling method using complementary oligonucleotide platform. Immunoblot analysis was performed on U-251 MG, U-87 MG, C6 and NCI-H460 cell lines to confirm the expression of tenascin-C. In vitro binding specificity of Tenascin-C aptamers was evaluated using confocal microscopy. In vivo biodistribution properties and PET Imaging of the 18F-labeled Tenascin-C were determined in C6 tumor-bearing nude mice. High levels of tenascin-C protein were detected in C6, U-251 MG. and U-87 MG cell lines. However, tenascin-C protein levels were undetectable in NCI-H460 cells. Confocal fluorescence microscopy images showed that Cy5-labeled Tenascin-C aptamer bound specifically to tenascin-C protein expressing cell lines including C6, U-251 MG and U-87 MG, however, not bound to NCI-H460 cells. Tumor uptake of 18F-labeled Tenascin-C aptamer was 1.64 ± 0.36 at 30 min after injection in C6 tumor xenografts. C6 tumors were clearly visualized by microPET imaging in a tumor-bearing mouse at 60 after injection. Tenascin-C aptamer was successfully labeled with F-18 labeled complementary oligonucleotide by base-pair hybridization. In vitro data showed that the cODN platform-hybridized Tenascin-C aptamer was selectively bound to target tumor cells. In addition, C6 tumor xenografts in mice were clearly visualized on microPET imaging. Our study demonstrated that Tenascin-C may be a useful target molecule for the imaging of malignant gliomas. Citation Format: Jun Young Park, Ye Lim Cho, Ju Ri Chae, Ga Eul Chu, Won Gil Cho, Sang Wun Kim, Won Jun Kang. Development and application of 18F-labeled Tenascin-C aptamer for cancer imaging [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4114.

Abstract 5676: Patient-derived tumor xenografts in humanized NSG-SGM3 mice: An improved immuno-oncology platform

Abstract The JAX® Onco-Hu® platform utilizes humanized mice engrafted with tumors to enable in vivo investigation of the interactions between the human immune system and human cancer. We have recently shown that humanized NOD-scid IL2Rγnull (NSG™) mice bearing patient-derived xenografts (PDX) allow efficacy studies of checkpoint inhibitors. A major avenue of our investigation is to generate murine humanized models containing a more complete human hematopoietic system and robust innate immune cell population. Next-generation NSG strains include triple transgenic NSG mice (NSG-SGM3) expressing myelosupportive human cytokines KITLG, CSF2, and IL-3. When engrafted with CD34+ human hematopoietic progenitor cells (HPCs) from CD3-depleted umbilical cord blood, NSG-SGM3 mice produce higher myeloid and Treg populations in the circulation as compared to NSG mice over 18 weeks post engraftment. We implanted an array of PDX tumors into humanized NSG-SGM3 mice at 2-3 months post engraftment. Tumors were dissociated and single-cell infiltrates were analyzed by multicolor flow cytometry with a focus on examining overall immune cell infiltration and the levels of hCD33+ myeloid cells. In the PS4050 melanoma PDX model, we found that hCD45+ cell infiltration was significantly increased in hu-NSG-SGM3 mice as compared to hu-NSG mice engrafted with the same HPC donor (3.7% vs. 1% of viable cells). The majority of tumor-infiltrating cells in hu-NSG-SGM3 mice expressed hCD33 (55% of hCD45+) and the percentage was significantly higher than that in hu-NSG mice (13%). hCD3+T cell infiltration level was similar between these two strains (~20% of hCD45+). PS4050-bearing hu-NSG-SGM3 mice treated with the anti-PD-1 antibody pembrolizumab (Keytruda) showed a significant reduction in tumor growth and the PD-1 levels in tumor-infiltrating T cells were greatly reduced by flow cytometry analysis. The overall hCD45+ cell infiltration and the frequencies of hCD4+T, hCD8+T, and hCD33+myeloid cells in tumors remained similar after treatment. Lastly, we observed that the effect of Keytruda on tumor growth reduction in hu-NSG-SGM3 mice is PD-L1-dependent using the human lung carcinoma cell line NCI-H460 depleted of PD-L1 expression by CRISPR. Keytruda treatment significantly reduced mock-transfected NCI-H460 cell growth. By comparison, PD-L1 KO NCI-H460 cells grew more slowly than the mock cells and lost the response to Keytruda. Together, these results indicate that PDX tumor-implanted hu-NSG-SGM3 mice serve as an important platform for understanding human immune system and tumor microenvironment interactions and for preclinical immuno-oncology efficacy studies. Citation Format: Li-Chin Yao, Mingshan Cheng, Ken-Edwin Aryee, Pooja Kumar, Nicole Walsh, Dale Greiner, Leonard Shultz, Edison T. Liu, Michael Brehm, James G. Keck. Patient-derived tumor xenografts in humanized NSG-SGM3 mice: An improved immuno-oncology platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5676.

Abstract 4788: Preclinical evaluation of E-191, a highly selective EGFR T790M inhibitor

Abstract Objective: Patients of non-small cell lung cancer (NSCLC) with activating EGFR mutations initially respond to the first generation reversible EGFR tyrosine kinase inhibitors, such as gefitinib and erlotinib. However, most of the patients acquire resistance within 10~16 months. The most common mechanism of resistance is the EGFR T790M mutation. In addition, selectivity of the EGFR inhibitors is important, as inhibition of EGFR wild type can lead to dose limiting toxicities, including rash and diarrhea. This may be the reason why the second generation irreversible EGFR inhibitors, such as afatinib, which also inhibit EGFRWT, is not widely used. Here, we disclose our clinical candidate compound E-191 as a novel irreversible inhibitor that potently inhibits the resistance mutant forms of EGFR with a good selectivity sparing EGFRWT. Method: The anti-proliferative activity of E-191 was evaluated in NCI-H1975 (EGFRT790M/L58R), HCC827 (EGFRd746-750), and A431 (EGFRWT) cell lines. Daily oral administration of E-191 at 3 doses was used to evaluate the in vivo anti-tumor activity in three cell-derived tumor xenograft (CDX) models, NCI-H1975, HCC827, and Ba/F3 (EGFRT790M /d746-750). The in vivo selectivity was evaluated in the A431 (EGFRWT) CDX model. Effects on EGFR signaling pathway were assessed by western blot analysis of the downstream effector proteins p-EGFR (Y1068), p-AKT (Ser473) and p-ERK (Thr202/Tyr204). Result: E-191 displayed potent anti-proliferative activity in the EGFR mutant cell lines NCI-H1975 (IC50 = 22 nM) and HCC827 (IC50=1.9 nM), and a much less potent activity in the EGFRWT A431 cell line (IC50 = 531 nM). E-191 has about 24 fold selectivity for EGFRT790M over EGFRWT. E-191 potently inhibited tumor growth at all doses tested (P&amp;lt;0.001) in the NCI-H1975, HCC827 and Ba/F3 (EGFRT790M /d746-750) models, while significantly less so in the A431 model. Western blot analysis of the tumor samples in the NCI-H1975 model study showed that E-191 inhibited p-EGFR (Y1068), p-AKT (Ser473), and p-ERK (Thr202/Tyr204), indicating that the tumor growth inhibition was through inhibition of the resistant mutant EGFR. Conclusion: We have demonstrated with in vitro and in vivo preclinical models that E-191 is a selective inhibitor of the resistance mutant forms of EGFR, potently inhibiting EGFRT790M but not EGFRWT. These results make us believe that E-191 has a good potential in the clinic for the treatment of resistant EGFR driven NSCLC. Citation Format: Liu Xile, Lihong Hu, Charles Z Ding, Weifeng Mao, Liqing Wang, Xiaohe Shi, Chaofeng Long, Xiaoxin Chen, Haijun Li, Xing Liu, Zhuowei Liu. Preclinical evaluation of E-191, a highly selective EGFR T790M inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4788.

Abstract 2563: Macrophage Toll-like receptor-chimeric antigen receptors (MOTO-CARs) as a novel adoptive cell therapy for the treatment of solid malignancies

Abstract Recent clinical trials using chimeric antigen receptors (CAR) T cells have demonstrated tremendous success in eradicating hematologic malignancies. Notwithstanding the excitement generated by CAR T cell therapy, its clinical efficacy has not been effectively translated to the context of solid tumors; the physical barriers of solid malignancies and the immunosuppressive conditions at the tumor site hinder the efficacy of CAR T cells. Macrophages have the ability to infiltrate almost every tissue and frequently are recruited into tumors. Therefore, macrophages are an attractive vehicle for CAR therapy and could help solve current challenges that CAR T cells face in the treatment of solid tumors. MOTO-CAR cells are monocyte-derived human macrophages that are genetically modified by a lentiviral or adenoviral approach to express a synthetic tumor-targeting receptor and to secrete cytokines, ligands or chemokine receptors. MOTO-CAR receptors are composed of a single-chain variable fragment (ScFv) that binds to a specific tumor target, a hinge to link it to a transmembrane domain, and an engineered Toll/Interleukin-1 receptor (TIR) signaling domain. When the ScFv binds to the tumor cell via its tumor target, an activation signal is transmitted. Myd88 dependent and independent signaling cascades are elicited, activating the macrophage and polarizing it towards a proinflammatory phenotype to eliminate cancer cells in a selective way. We previously reported the expression of Thymidine Kinase 1 (TK1) on the cell membrane of the non-small cell lung carcinoma NCI-H460 and A549 cell lines. The in vitro function of TK1 MOTO-CAR cells was evaluated against these cancer cell lines, using GFP-based phagocytosis and killing assays. Additionally, cell migration and interaction was recorded using time-lapse video with a confocal microscope. Upon co-culturing, with its target TK1 specific MOTO-CARs showed a nearly 4-fold increase in killing activity when compared with the controls (p&amp;lt;0.01). MOTO-CAR cells were produced through a lentiviral approach with around 30% of the cells expressing MOTO-CARs and with an adenoviral approach using the Ad5f35 vector with an efficiency of 70-80% of cells being transduced. Furthermore, after transduction MOTO-CAR cells showed a consistent M1 phenotype expressing high levels of CD14, CD80, CD206 and low levels of CD163. Time-lapse videos showed migration and clustering of MOTO-CAR cells around H460 GFP + cells. Moreover, cell death was observed upon contact of MOTO-CAR cells with target cells as well as phagocytic activity. In vivo testing using an orthotopic NOD scid gamma mice model is in progress. Our preclinical data show evidence that human macrophages are a suitable vehicle for CAR therapy and have the potential to successfully extrapolate the clinical efficacy of CAR therapy to the context of solid tumors. Citation Format: Edwin J. Velazquez, John E. Lattin, Taylor D. Brindley, Zachary Z. Reinstein, Roger Chu, Lu Liu, Evita G. Weagel, Michelle H. Townsend, Kiara V. Whitley, Eliza L. Lawrence, Brandon T. Garcia, Scott Weber, Richard A. Robison, Kim L. O'Neill. Macrophage Toll-like receptor-chimeric antigen receptors (MOTO-CARs) as a novel adoptive cell therapy for the treatment of solid malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2563.

Ligand-based pharmacophore model for the discovery of novel CXCR2 antagonists as anti-cancer metastatic agents.

Metastatic cancer is considered a fatal progression of cancer worldwide. It has been shown that a key player in this scenario is the CXC chemokine receptor 2 (CXCR2). To identify novel CXCR2 antagonists, a pharmacophore model was built with the HipHop program by screening a database containing compounds which were designed based on the known structure–activity relationship (SAR) of the diarylurea series CXCR2 antagonists. Compound 1a bearing the novel skeleton was selected from database screening and subjected to the in vitro biological test which showed a moderate CXCR2 antagonist potential. With further modification and exploration of SAR, compound 1e demonstrated improved CXCR2 antagonist activity with an IC50 value of 14.8 µM. Furthermore, wound healing assay using the NCI-H1299 cell line indicated that 1e showed an excellent anti-cancer metastatic effect (72% inhibition in cell migration at 50 µg ml−1).

Abstract 1170: NCI-H1975 L858R/T790M/C797S isogenic cell line for next-generation EGFR inhibitor screening

Abstract Non-small-cell lung cancer (NSCLC) is the most common cause of cancer death. Over-activation of EGFR occurs in 13%~23% of NSCLC. The identification of EGFR as a cancer driver gene had led to rapid development of NSCLC targeted therapy, but the mutation of EGFR during treatment can cause drug resistance and cancer relapse. For example, T790M mutation causes resistance to the first generation of EGFR inhibitor Erlotinib and C797S mutation contributes to the resistance to the 3rd EGFR inhibitor AZD9291. There are several human cell lines for T790M mutation research, but there is not yet any primary human cancer cell line with C797S mutation. In the current study, a human isogenic EGFR C797S mutation cell line was generated through introducing nucleotide mutation into the EGFR gene in NCI-H1975 cell line using CRISRP technology, which originally bears L858R and T790M mutations of EGFR. First, the sgRNA sequence around the exon-20 of EGFR gene was screened using CRIPSR plasmid. Second, the donor plasmid with the homology arm containing exon-20 of EGFR and selecting marker was constructed and co-transfected into the parental cell line. Then, the NCI-H1975 C797S isogenic cell line was obtained and confirmed after screening over 100 single clones using the PCR method. The NCI-H1975 C797S isogenic cell line was resistant to AZD9291 but sensitive with EAI045 (the potential 4th generation EGFR inhibitor) combined with cetuximab. These data indicate that our NCI-H1975 L858R/T790M/C797S isogenic line is a useful model for next generation EGFR inhibitor study. Citation Format: Feng Hao, Feng He, Wenna Zhang, Zhaoshuai Bai, Jinying Ning. NCI-H1975 L858R/T790M/C797S isogenic cell line for next-generation EGFR inhibitor screening [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1170.

Abstract 4794: Preclinical evaluation of WX-0593, a potent orally active ALK inhibitor

Abstract Objective: ALK is a tyrosine kinase belonging to the insulin receptor super-family. The constitutively active ALK fusions are oncogenic and linked with multiple types of human cancer. The EML4-ALK fusion has been proven to be the driver of approximately 5% of NSCLC patients. We disclose here for the first time a novel ALK inhibitor WX-0593 and its preclinical evaluation data in preclinical models of ALK driven NSCLC. Method: The enzyme activities of WX-0593 for ALKWT, ALKL1196M, ALKC1156Y and EGFRL858R/T790M were measured with corresponding kinase assays. The anti-proliferative activity was evaluated in Karpas-299, Ba/F3, Ba/F3 (EML4-ALK-WT), Ba/F3 (EML4-ALK-L1196M), Ba/F3 (EML4-ALK-C1156Y), and NCI-H1975 cell lines. The specific effect (PD) of WX-0593 on ALK signaling was analyzed in NCI-H3122 cells. Antitumor activity of WX-0593 was evaluated in three patient-derived xenograft (PDX) models of NSCLC (LU-01-0015, LU-01-0319, and crizotinib resistant LU-01-0319R) and one cell-derived xenograft (CDX) model of NSCLC (NCI-H3122). PKPD was assessed by western blot analysis of the treated tumor tissues for p-ALK (Tyr1604), p-STAT3 (Tyr705), p-STAT5 (Tyr694), p-AKT, and p-ERK. Result: WX-0593 displayed potent enzymatic activities for ALKWT, ALKL1196M, ALKC1156Y and EGFRL858R/T790M with IC50 of 5.38, 9.26, 9.28, and 16.74 nM respectively. It also showed expected anti-proliferative activity in six cell lines, Karpas-299, Ba/F3, Ba/F3 (EML4-ALK-WT), Ba/F3 (EML4- ALK-L1196M), Ba/F3 (EML4-ALK-C1156Y), and NCI-H1975, with IC50 of 4.5, 2208, 4, 9.5, 9, and 508 nM, respectively. Western blot study in NCI-H3122 cells showed that WX-0593 could fully inhibit p-ALK, p-ERK,p-STAT5 at 11.1 nM and p-AKT at 100 nM. WX-0593 significantly inhibited tumor growth at all doses tested (P&amp;lt;0.001) in the LU-01-0015 (HIP1-ALK), LU-01-0319 (EML4-ALK), and LU-01-0319R NSCLC PDX models as well as in the NCI-H3122 CDX model. Western blot analysis of the tumor samples showed that WX-0593 inhibited p-ALK (Tyr1604), p-STAT3 (Tyr705), p-STAT5 (Tyr694), p-AKT, and p-ERK, indicating that the tumor growth inhibition was mediated by inhibition of ALK signaling. Conclusion: We have identified a potent orally active second generation ALK inhibitor, WX-0593. It can inhibit the activity of both wide type and crizotinib resistant ALK and showed strong antitumor activity in in vitro and in vivo preclinical models. These results are considered highly promising and warrant moving the compound forward to clinical investigation. Citation Format: Liu Xile, Charles Z Ding, Weifeng Mao, Yuxin Qin, Shansong Zheng, Yingying Yang, Qingmei Zheng, Long Zhang, Shuyong Zhao. Preclinical evaluation of WX-0593, a potent orally active ALK inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4794.

Abstract 485: Modulation of microRNA expression in EGFR-tyrosine kinase inhibitor-resistant non-small cell lung cancer cell lines

Abstract Objectives: Epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) are now standard of care in lung cancer patients with EGFR mutation. Invariably, these patients develop resistance and require treatment with another EGFR-TKI or chemotherapy. The gatekeeper mutation T790M is responsible for about 50% of resistance. Other mechanisms may also confer resistance in these patients. High expression of microRNA-10b (miRNA-10b) is associated with worse prognosis in resected lung cancer patients with EGFR mutation. Both miRNA-27a and -27b are associated with increased expression of c-met, which is a potential mechanism of resistance to EGFR-TKI. The goal of this study is to determine the optimal treatment and miRNA characteristic of EGFR mutation lung cancer cells resistant to EGFR-TKI. We hypothesized that miRNA-10b, -27a and -27b modulated Erlotinib (ERL) resistance by different EGFR-TKIs and chemotherapeutic agents in EGFR mutant NSCLC cell lines. Materials and Methods: Lung cancer cell lines with EGFR mutation HCC827 and HCC4006 resistant to ERL were generated. ERL-resistant cells were then treated with 10 nM afatinib (Afa), 0.5 mg/mL pemetrexed (Pem), and combination of 10 nM Afa/0.5mg/mL Pem for 72h. The cells were harvested and microRNA assay was performed by real-time PCR. NCI-H460 cell line without EGFR mutation was used as control. Results: We observed increased expression of miRNA-10b, miRNA-27a and miRNA-27b in ERL-resistant HCC4006 compared to control cells NCI-H460. There was an upregulation of miRNA-27a and -27b but not of miRNA-10b in ERL-resistant HCC827. Treatment of ERL-resistant cells with Afa, Pem, and combined treatment changed the expression of miRNA-10b, miRNA-27a and miRNA-27b. These miRNAs were downregulated by 2nd-generation EGFR-TKI therapy alone or in combination with Pem. Different treatments had different effects on these miRNAs. There might be differences in miRNA regulation in different cell lines when treated with EGFR-TKI or chemotherapy. Our results suggest that microRNA may be used as targets for developing novel approach to treating patients with EGFR-TKI resistance. Conclusion: This study may have implications in the management of patients resistant to EGFR TKI. MicroRNA analysis showed upregulation of several microRNAs that should be confirmed in clinical setting. Citation Format: Inamul Haque, Hannah Motes, Mukut Sharma, Emma Borrego-Diaz Reyes, Andrew K. Godwin, Chao H. Huang. Modulation of microRNA expression in EGFR-tyrosine kinase inhibitor-resistant non-small cell lung cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 485.

Jizanpeptins, Cyanobacterial Protease Inhibitors from a Symploca sp. Cyanobacterium Collected in the Red Sea.

Jizanpeptins A-E (1-5) are micropeptin depsipeptides isolated from a Red Sea specimen of a Symploca sp. cyanobacterium. The planar structures of the jizanpeptins were established using NMR spectroscopy and mass spectrometry and contain 3-amino-6-hydroxy-2-piperidone (Ahp) as one of eight residues in a typical micropeptin motif, as well as a side chain terminal glyceric acid sulfate moiety. The absolute configurations of the jizanpeptins were assigned using a combination of Marfey's methodology and chiral-phase HPLC analysis of hydrolysis products compared to commercial and synthesized standards. Jizanpeptins A-E showed specific inhibition of the serine protease trypsin (IC50 = 72 nM to 1 μM) compared to chymotrypsin (IC50 = 1.4 to >10 μM) in vitro and were not overtly cytotoxic to HeLa cervical or NCI-H460 lung cancer cell lines at micromolar concentrations.

Novel carbamate-linked quaternary ammonium lipids containing unsaturated hydrophobic chains for gene delivery

In this paper, two novel carbamate-linked quaternary ammonium lipids (MU18: a lipid with a mono-ammonium head; GU18: a lipid with a Gemini-ammonium head) containing unsaturated hydrophobic chains were designed and synthesized. The chemical structures of the synthetic lipids were characterized by infrared spectrum, ESI-MS, 1H NMR, 13C NMR, and HPLC. For investigating the effect of unsaturation on gene delivery, the previous reported saturated cationic liposomes (MS18 and GS18) were used as comparison. Cationic liposomes were prepared by using these cationic lipids and neutral lipid DOPE at the molar ratio of 1:1. Particle sizes and zeta potentials of the cationic liposomes were studied to show that they were suitable for gene transfection. The binding abilities of the cationic liposomes were investigated by gel electrophoresis at various N/P ratios from 0.5/1 to 8/1. The results indicated that the binding ability of GU18 was much better than MU18 and the saturated cationic liposomes (MS18 and GS18). DNA transfection of these liposomes comparable to commercially available reagent (DOTAP) was achieved in vitro against Hela, HepG-2 and NCI-H460 cell lines. GU18 showed higher transfection at the N/P ratio of 3/1 than other cationic liposomes and the positive control, DOTAP. All of the liposomes presented a relatively low cytotoxicity, which was measured by MTT. Therefore, the synthetic lipids bearing unsaturated hydrophobic chains and Gemini-head could be promising candidates for gene delivery.

P-326 - Eucalyptus globulus Labill. decoction extract inhibits the growth of human tumor cell lines

Eucalyptus globulus Labill. is a widespread evergreen plant with a plethora of medicinal properties, most notably antitumoral activity. The aims of this work were to: (i) test the cell growth inhibitory effect of the E. globulus, prepared by decoction and infusion, in three human tumor cell lines models: lung (NCI-H460), colorectal (HCT-15) and pancreatic (PANC-1) cancer; (ii) study the mechanism of action of the most potent extract in the most sensitive cell line. Both extracts inhibited the growth of all cell lines and revealed gallotannins as the main compounds. The decoction extract was the most potent in inhibiting the NCI-H460 cell growth. Thus, we further investigated the effect of the decoction extract on the NCI-H460 cell viability (Trypan blue exclusion assay), cell cycle profile, apoptosis (Flow cytometry) and protein expression (Western blot). Interestingly, the E. globulus decoction extract caused a dose-dependent decrease in cell number, which was correlated with a cell cycle arrest in the G1 phase and an increase in p53 and p21 proteins expression. However, no differences were found in the levels of programmed cell death. The relevance of identifying novel bioactive compounds with antitumoral activity in Eucalyptus globulus L. is thus highlighted in this work.

Chemical constituents from Kalanchoe hybrida and their cytotoxicity

ObjectiveKalanchoe hybrida (Crassulaceae) is naturalized throughout all the island of Taiwan in China. The preliminary bioassay-guided fractionation of the crude extract of K. hybrida exhibited that the chloroform and n-butanol fractions possessed potent cytotoxicity against MCF-7, NCI-H460, and SF-268 tumor cell lines at 50 µg/mL concentration. Therefore, K. hybrida was selected as a target and the chemical constituents from the chloroform and n-butanol fractions of the crude extracts of K. hybrida were identified. The potential constituents were examined for their cytotoxicity against the tumor cell lines. MethodsA combination of conventional chromatographic techniques was performed on the crude extract of K. hybrida. The chemical structures of the purified constituents were identified on the basis of spectroscopic and spectrometric analysis. ResultsThe purification results had led to the characterization of totally 37 compounds. The isolated compounds 1, 2, and 4–12 were examined for their cytotoxicity in vitro, and bufadienolides 4–8 and flavonol glycoside 11 displayed significant cytotoxicity towards all the tested tumor cell lines among these tested compounds. ConclusionThe results indicated that these principles should be responsible for the bioactivity of corresponding partial fractions. The potential constituents could be further investigated to explore the new natural lead drugs.

Novel macamides from maca (Lepidium meyenii Walpers) root and their cytotoxicity

Maca, Lepidium meyenii Walpers (Brassicaceae), contains secondary metabolites, including macamides, which possess many bioactive properties. Two new macamides, namely, N-benzyl-9-oxo-10E,12E-octadecadienamide (3) and N-benzyl-9-oxo-10E,12Z-octadecadienamide (4), were isolated from maca root. In addition, two fatty acid derivatives, 9-oxo-10E,12E-octadecadienoic acid (1) and 9-oxo-10E,12Z-octadecadienoic acid (2), were found for the first time from maca. The structures of compounds (3) and (4) were elucidated by spectrometric and spectroscopic methods including UV, IR, 1H NMR, 13C NMR, 2D NMR and HR ESI–MS experiments. The novel macamides exhibit inhibitory activity against the proliferation of the HT-29 cancer cell line with IC50 values of 12.8 μmol/L (3) and 5.7 μmol/L (4). The two compounds were inactive (IC50 >50 μmol/L) towards the proliferation of SGC7901, MCF7, NCI-H460 and HepG2 cancer cell lines.

A family of manganese complexes containing heterocyclic-based ligands with cytotoxic properties

We describe the synthesis of three new manganese (II) complexes containing the bidentate ligands 2-(1-methyl-3-pyrazolyl)pyridine (pypz-Me) and ethyl 2-(3-(pyridine-2-yl)-1H–pyrazol-1-yl)acetate (pypz-CH2COOEt), with formula [MnX2(pypz-Me)2] (X = Cl−1, CF3SO3−2) and [Mn(CF3SO3)2(pypz-CH2COOEt)2] 3. Complexes 1–3 have been characterized through analytical, spectroscopic and electrochemical techniques, as well as by monocrystal X-ray diffraction analysis. The complexes show a six-coordinated Mn(II) ion though different stereoisomers have been isolated for the three compounds. Complexes 1–3, together with the previously described compounds [MnCl2(pypz-H)2] 4, [Mn(CF3SO3)2(pypz-H)2] 5, [Mn(NO3)2(pypz-H)2] 6, [MnCl2(H2O)2(pypz-H)2] 7 (pypz-H = 2-(3-pyrazolyl)pyridine) and ([Mn(CF3SO3)2((−)-L)2] 8, ((−)-L = (−)-pinene[5,6]bipyridine), were tested in vitro for cytotoxic activity against NCI-H460 and OVCAR-8 cancer cell lines. The geometry of a specific compound does not seem to influence its activity in a significant extent. However, among the tested compounds those that display hydrophobic substituents on the pyrazole ring and triflate ions as labile ligands show the best antiproliferative properties. Specifically, compound 8 containing the pinene-bipyridine ligand presents an antiproliferative activity similar to that of cisplatin and higher than that of carboplatin, and displays selectivity for tumour cells. Its antiproliferative effect is due to the generation of ROS species that allow the compound to interact with DNA.

Synthesis and Biological Evaluation of Calothrixins B and their Deoxygenated Analogues.

A series of calothrixin B (2) analogues bearing substituents at the 'E' ring and their corresponding deoxygenated quinocarbazoles lacking quinone unit were synthesized. The cytotoxicities of calothrixins 1, 2, and 15b-p and quinocarbazole analogues were investigated against nine cancer cell lines. The quinocarbazoles 21a and 25a inhibited the catalytic activity of human topoisomerase II. The plasmid DNA cleavage abilities of calothrixins 1, 2, and 15b-p identified compound 15h causing DNA cleavage comparable to that of calothrixin A (1). Calothrixin A (1), 3-fluorocalothrixin 15h and 4-fluoroquinocarbazole 21b induced extensive DNA damage followed by apoptotic cell death. Spectral and plasmid unwinding studies demonstrated an intercalative mode of binding for quinocarbazoles. We identified two promising drug candidates, the 3-fluorocalothrixin B 15h with low toxicity in animal model and its deoxygenated derivative 4-fluoroquinocarbazole 21b as having potent cytotoxicity against NCI-H460 cell line with a GI50 of 1 nM.

A novel cyclohexenone from Trachyspermum roxburghianum

A novel cyclohexenone, roxydienone (1), together with 13 known compounds (2–14), were isolated from the seeds of Trachyspermum roxburghianum. The structures of these compounds were established on the basis of their 1D and 2D NMR spectroscopic data. Compounds 1, 2, 7–9 and 14 showed cytotoxicity against NCI-H187 cell line while compounds 1 and 9 showed cytotoxicity against KB cell line. In addition, only compound 13 showed cytotoxicity against MCF-7 cell line.