Cultured Tumor Cell Lines Research Articles

Synthesis, structural study and antitumor activity of novel alditol-based imidazophenanthrolines (aldo-IPs)

A series of 1H-imidazo [4,5-f][1,10] phenanthroline derivatives functionalized at 2-position with chiral, and conformationally flexible polyhydroxy alkyl chains derived from carbohydrates (alditol-based imidazophenanthrolines, aldo-IPs) is presented herein. These novel glycomimetics showed relevant and differential cytotoxic activity against several cultured tumor cell lines (PC3, HeLa and HT-29), dependent on the nature and stereochemistry of the polyhydroxy alkyl chain. The mannose-based aldo-IP demonstrated the higher cytotoxicity in the series, substantially better than cisplatin metallo-drug in all cell lines tested, and better than G-quadruplex ligand 360A in HeLa and HT29 cells. Cell cycle experiments and Annexin V-PI assays revealed that aldo-IPs induce apoptosis in HeLa cells. Initial study of DNA interactions by DNA FRET melting assays proved that the aldo-IPs produce only a slight thermal stabilization of DNA secondary structures, more pronounced in the case of quadruplex DNA. Viscosity titrations with CT dsDNA suggest that the compounds behave as DNA groove binders, whereas equilibrium dialysis assays showed that the compounds bind CT with Ka values in the range 104–105 M−1. The aldo-IP derivatives were obtained with synthetically useful yields through a feasible one-pot multistep process, by aerobic oxidative cyclization of 1,10‐phenanthroline‐5,6‐diamine with a selection of unprotected aldoses using (NH4)2SO4 as promoter.

Novel conditionally replicating adenovirus-mediated efficient detection of circulating tumor cells in lung cancer patients.

Circulating tumor cells (CTCs) are present in the blood of cancer patients from the early stage of cancer development, and their presence has been correlated with patient prognosis and treatment responses. Accordingly, CTCs have been attracting attention as a novel biomarker for early detection of cancer and monitoring of treatment responses. However, since patients typically have only a few CTCs per milliliter of blood, development of an accurate and highly sensitive CTC detection method is crucial. We previously developed a CTC detection method using a novel conditionally replicating adenovirus (Ad) that expresses green fluorescence protein (GFP) in a tumor cell-specific manner by expressing the E1 gene using a tumor-specific human telomerase reverse transcriptase (hTERT) promoter (rAdF35-142T-GFP). CTCs were efficiently detected using rAdF35-142T-GFP, but GFP expression levels in the CTCs and production efficiencies of rAdF35-142T-GFP were relatively low. In this study, in order to overcome these problems, we developed four types of novel GFP-expressing conditionally replicating Ads and examined their ability to visualize CTCs in the blood samples of lung cancer patients. Among the four types of novel recombinant Ads, the novel conditionally replicating Ad containing the 2A peptide and the GFP gene downstream of the E1A gene and the adenovirus death protein (ADP) gene in the E3 region (rAdF35-E1-2A-GFP-ADP) mediated the highest number of GFP-positive cells in the human cultured tumor cell lines. Titers of rAdF35-E1-2A-GFP-ADP were significantly higher (about 4-fold) than those of rAdF35-142T-GFP. rAdF35-E1-2A-GFP-ADP and rAdF35-142T-GFP efficiently detected CTCs in the blood of lung cancer patients at similar levels. GFP+/CD45- cells (CTCs) were found in 10 of 17 patients (58.8%) for both types of recombinant Ads.

JSKN003, A NOVEL BIPARATOPIC ANTI-HER2 ANTIBODY-DRUG CONJUGATE, EXHIBITS POTENT ANTITUMOR EFFICACY

Abstract In human advanced solid tumors, expression of HER2 protein has been reported in various tumor tissues and a variety of cultured tumor cell lines including breast cancer, gastric cancer, pancreatic cancer, lung cancer, colorectal cancer, and ovarian cancer. Due to the critical roles of HER2 in carcinogenesis, two main targeted therapies have been developed in the past two decades to block the HER2-driven pathways, which include small molecule compounds that inhibit the tyrosine kinase activity of the intracellular domain, and mono-antibodies (mAbs) that target the extracellular domain (ECD) of the receptors. JSKN003 is an antibody-drug conjugate (ADC) comprised of a recombinant, humanized anti-human epidermal growth factor receptor 2 (HER2) bispecific antibody conjugated to a topoisomerase I inhibitor via a dibenzocyclooctyne tetrapeptide linker. The anti-HER2 component, KN026, is a recombinant, humanized bispecific antibody that targets both extra-cellular domains II (pertuzumab binding site) and IV (trastuzumab binding site) of HER2. JSKN003 showed high affinity binding to human HER2 with KDs of 2.209 E-10M, which is comparable to its parental antibody KN026 and bound to NCI-N87 and BxPC-3 cells in a concentration-dependent manner. At the same time JSKN003 showed more extensive and faster internalization than DS8201 on NCI-N87 cells. As expected, JSKN003 showed directly inhibits growth by targeting HER2 positive tumor model (NCI-N87 and BT474 cell models). The single dose and multiple dose pharmacokinetics study in cynomolgus monkey indicated that JSKN003, total antibody and DXd had general linear dynamic characteristics, and pharmacokinetics parameters showed no significant differences between males and females in the range 0.3-30 mg/kg. The HNSTD (highest non-severely toxic dose) of JSKN003 was determined as 30mg/kg in cynomolgus monkeys. These preclinical data suggest that JSKN003 could potentially benefit patients with tumors co-expressing HER2 through improved drug selectivity and efficacy. JSKN003’s safety, tolerability and preliminary anti-tumor activity are currently being evaluated in a first-in-human phase I study in advanced stage solid tumors in Australia (NCT05494918) using a BOIN design. This ADC is targeted on the subjects who has HER2 expression and/or HER2-gene mutation and may address an unmet medical need for these patients.

Novel method for highly multiplexed gene expression profiling of circulating tumor cells (CTCs) captured from the blood of women with metastatic breast cancer

BackgroundEnumeration of circulating tumor cells (CTCs) has proven clinical significance for monitoring patients with metastatic cancers. Multiplexed gene expression profiling of CTCs is a potential tool for assessing disease status and monitoring treatment response. The Parsortix® technology enables the capture and harvest of CTCs from blood based on cell size and deformability. The HyCEAD™ (Hybrid Capture Enrichment Amplification and Detection) assay enables simultaneous amplification of short amplicons for up to 100 mRNA targets, and the Ziplex™ instrument quantifies the amplicons for highly sensitive gene expression profiling down to single cell levels. The aim of the study was to functionally assess this system.MethodsThe HyCEAD/Ziplex platform was used to quantify the expression levels for 72 genes using as little as 20 pg of total RNA or a single cultured tumor cell. Assay performance was evaluated using cells or total RNA spiked into Parsortix harvests of healthy donor blood. The assay was also evaluated using total RNA obtained from Parsortix harvests of blood from metastatic breast cancer (MBC) patients or healthy volunteers (HVs).ResultsUsing genes with low expression in WBC RNA and/or in unspiked Parsortix harvests from HVs, the assay distinguished between the different breast cancer and ovarian cancer cell lines with as little as 20 pg of total RNA (equivalent to a single cell) in the presence of 1 ng of WBC RNA. Single cultured cells spiked into Parsortix harvests from 10 mL of HV blood were also detected and distinguished from each other. CVs from repeatability experiments were less than 20%. Hierarchical clustering of clinical samples differentiated most MBC patients from HVs.ConclusionHyCEAD/Ziplex provided sensitive quantification of expression of 72 genes from 20 pg of total RNA from cultured tumor cell lines or from single cultured tumor cells spiked into lysates from Parsortix harvests of HV blood. The HyCEAD/Ziplex platform enables the quantification of selected genes in the presence of residual nucleated blood cells in Parsortix harvests. The HyCEAD/Ziplex platform is an effective tool for multiplexed molecular characterization of mRNA in small numbers of tumor cells harvested from blood.

Accumulation of STR-Loci Aberrations in Subclones of Jurkat Cell Line as a Model of Tumor Clonal Evolution

Many genetic markers are known to distinguish tumor cells from normal. Genetic lesions found at disease onset often belong to a predominant tumor clone, and further observation makes it possible to assess the fate of this clone during therapy. However, minor clones escape monitoring and become unidentified, leading to relapses. Here we report the results of in vitro study of clonal evolution in cultured tumor cell line (Jurkat) compared to the cell line of non-tumor origin (WIL2-S). Cell lines were cultured and cloned by limiting dilutions. Subclones were tested by short tandem repeats (STR) profiling. Spontaneous STR aberrations in cells of non-tumor origin occur in less than 1 of 100 cultured cells. While in the cells of tumor origin, new aberrations appear in 1 or even more of 3 cultured cells. At the same time, a significant relationship was found between the accumulation of aberrations in the pool of subclones and the rate of cell growth. One can speculate that this approach could be applied for the analysis of primary patient tumor cell culture to obtain information concerning the evolutionary potential of the tumor cells that may be useful for the selection of a therapy approach.

Optimal timing of a γH2AX analysis to predict cellular lethal damage in cultured tumor cell lines after exposure to diagnostic and therapeutic radiation doses.

Phosphorylated H2AX (γH2AX) is a sensitive biomarker of DNA double-strand breaks (DSBs). To assess the adverse effects of low-dose radiation (<50mGy), γH2AX levels have typically been measured in human lymphocytes within 30min of computed tomography (CT) examinations. However, in the presence of DSB repair, it remains unclear whether γH2AX levels within 30min of irradiation completely reflect biological effects. Therefore, we investigated the optimal timing of a γH2AX analysis to predict the cell-surviving fraction (SF). Three tumor cell lines were irradiated at different X-ray doses (10-4000mGy), and the relationships between SF and relative γH2AX levels were investigated 15min and 2, 6, 12 and 24h after irradiation. Data were analyzed for high-dose (0-4000mGy) and low-dose (0-500mGy) ranges. Correlations were observed between SF and the relative number of γH2AX foci/nucleus at 12h only (R2 = 0.68, P = 0.001 after high doses; R2 = 0.37, P = 0.016 after low doses). The relative intensity of γH2AX correlated with SF 15min to 12h after high doses and 2 to 12h after low doses, with the maximum R2 values being observed 2h after high doses (R2 = 0.89, P < 0.001) and 12h after low doses (R2 = 0.65, P < 0.001). Collectively, cellular lethal damage in tumor cells was more accurately estimated with residual DSBs 12h after low-dose (10-500mGy) irradiation. These results may contribute to determination of the optimal timing of biodosimetric analyses using γH2AX in future studies.

Untouched isolation enables targeted functional analysis of tumour-cell-derived extracellular vesicles from tumour tissues.

To accurately identify the functions of tumour‐cell‐derived extracellular vesicles (T‐EVs), EVs directly isolated from tumour tissues are much preferred over those derived from in vitro cultured tumour cell lines. However, the functional analysis of T‐EVs has still been severely limited by the difficulty in selective isolation of T‐EVs from tissue‐derived heterogeneous EVs, which also contain non‐tumour cell‐derived EVs. We here establish an untouched isolation strategy that specifically collects natural T‐EVs from tumour tissues by removing non‐tumour‐cell‐derived EVs. Different from traditional immunomagnetic separation, our isolation materials are directly bound to undesired non‐tumour‐cell‐derived EVs, preserving the natural properties of T‐EVs. Using this strategy, we reveal the distinct performances of tissue‐derived T‐EVs in organotropism to lymph nodes, immunosuppression and angiogenesis. The present work, which takes an extraordinary step forward in the isolation of EV subpopulation from tumour tissues, would dramatically accelerate the investigation of EV heterogeneity.

Novel Anthracycline Utorubicin for Cancer Therapy.

Novel anticancer compounds and their precision delivery systems are actively developed to create potent and well‐tolerated anticancer therapeutics. Here, we report the synthesis of a novel anthracycline, Utorubicin (UTO), and its preclinical development as an anticancer payload for nanocarriers. Free UTO was significantly more toxic to cultured tumor cell lines than the clinically used anthracycline, doxorubicin. Nanoformulated UTO, encapsulated in polymeric nanovesicles (polymersomes, PS), reduced the viability of cultured malignant cells and this effect was potentiated by functionalization with a tumor‐penetrating peptide (TPP). Systemic peptide‐guided PS showed preferential accumulation in triple‐negative breast tumor xenografts implanted in mice. At the same systemic UTO dose, the highest UTO accumulation in tumor tissue was seen for the TPP‐targeted PS, followed by nontargeted PS, and free doxorubicin. Our study suggests potential applications for UTO in the treatment of malignant diseases and encourages further preclinical and clinical studies on UTO as a nanocarrier payload for precision cancer therapy.

Novel Anthracycline Utorubicin for Cancer Therapy.

Novel anticancer compounds and their precision delivery systems are actively developed to create potent and well-tolerated anticancer therapeutics. Here, we report the synthesis of a novel anthracycline, Utorubicin (UTO), and its preclinical development as an anticancer payload for nanocarriers. Free UTO was significantly more toxic to cultured tumor cell lines than the clinically used anthracycline, doxorubicin. Nanoformulated UTO, encapsulated in polymeric nanovesicles (polymersomes, PS), reduced the viability of cultured malignant cells and this effect was potentiated by functionalization with a tumor-penetrating peptide (TPP). Systemic peptide-guided PS showed preferential accumulation in triple-negative breast tumor xenografts implanted in mice. At the same systemic UTO dose, the highest UTO accumulation in tumor tissue was seen for the TPP-targeted PS, followed by nontargeted PS, and free doxorubicin. Our study suggests potential applications for UTO in the treatment of malignant diseases and encourages further preclinical and clinical studies on UTO as a nanocarrier payload for precision cancer therapy.

Scanning Proton FLASH Irradiation Using a Synchrotron Accelerator: Effects on Cultured Cells and Differences by Irradiation Positions

An ultra-high dose rate radiation (FLASH) is now attracting great attention. Although FLASH radiation with a cyclotron-type proton therapy machine has been reported, production of the ultra-high dose rate using other machines has scarcely been reported. In this study, we established a FLASH radiation system using a synchrotron proton beam accelerator and compared the effects of FLASH and conventional dose-rate radiation on cultured cells. Both types of radiation were delivered at the 1-cm spread-out Bragg peak (SOBP) and plateau region before SOBP. Protons were accelerated by adjusting the application pattern of high-frequency power. Other incident conditions for passive irradiation were also applied to increase proton accumulation to approximately twice the normal scanning conditions. Ultra-high dose rates were achieved by applying a high-frequency power dozens of times higher than usual to extract all protons within approximately 50 ms, thereby monitoring with a Faraday cup immediately after synchrotron extraction and adjusting the waveform. The dose and dose rates were measured using a float glass, gafchromic film, and plane parallel ionization chamber. Four cultured tumor cell lines and two normal cell lines were used. FLASH radiation at 6 and 10 Gy (200 Gy/s, 1 shot) and conventional radiation at 6 and 10 Gy (3 Gy/s) were delivered to the SOBP and plateau region before SOBP. After radiation, appropriate amounts of reagents to stain caspase-3/7 were added, and differences in cell growth rates and cell cycle were examined using live-cell fluorescence time-lapse imaging. The cell confluence ratio and apoptotic change were also quantified. V79 spheroids were subjected to FLASH (300 Gy/s, 10 and 18 Gy, 1 shot) and conventional radiation (3 Gy/s, 10 and 18 Gy) at SOBP. Subsequently, they were disaggregated to single cells and assayed for cell survival by colony assay. A 1-cm SOBP was formed by a mini-ledge filter was prepared with a high-definition 3D printer. FLASH radiation could be achieved at 40-800 Gy/s. In vitro, similar cell-killing effects were observed for FLASH and conventional radiation in the cancer cells, with cell-cycle delay. Significant apoptotic effects and G2 arrest during the cell cycle were observed for FLASH peak irradiation, probably due to the higher LET at the Bragg peak. Decreased proliferation and cell-killing effects were also observed in the normal cells for FLASH, but the apoptotic effect was not different between FLASH and conventional irradiation. Stronger cell-killing effects were observed with the V79 spheroids for FLASH peak than conventional peak irradiation. Scanning proton FLASH radiation with a synchrotron accelerator was established for biological studies. FLASH seemed to be equally effective for cancer and normal cells in vitro. The effects were higher with FLASH peak irradiation owing to the high LET at the Bragg peak, which is a characteristic of synchrotron accelerators.

Abstract 6272: Redirecting tumor extracellular proteases to target radiosensitizer delivery

Abstract Patients with locally advanced cancers are treated with combined radiotherapy and chemotherapy, however curability is poor and side effects severe. Drugs that sensitize tumors to radiotherapy have been tried to improve cure, but tumor specificity remains a challenge. To achieve tumor selectivity of small molecule radiosensitizers, we tested as a strategy active tumor targeting utilizing peptide-based drug conjugates that split the responsibilities for targeting and radiosensitization into distinct molecular tasks. The tumor milieu possesses localizing beacons that can guide drug delivery using tumor cell surface receptors and microenvironment extracellular matrix metalloproteinases (MMPs). Here we applied peptide-based linkers to test for tumor selective drug delivery and radiosensitization by chemically conjugating the DNA damage checkpoint kinase 1/2 inhibitor AZD7762 to antibody to or cell penetrating peptides. AZD7762 governs DNA damage response and irradiated cell fate. We synthesized drug conjugates of AZD7762 and compared their anti-tumor efficacy in combination with ionizing radiation (IR). An antibody drug conjugate (ADC) was built by attaching AZD7762 to the anti-EGFR antibody cetuximab. Polycationic cell penetrating peptide drug conjugates were constructed by linking AZD7762 to a 9 amino acid repeat of D-arginine and keeping it bare (r9-AZD7762) or cloaking r9 within a MMP-2/9 triggered activatable cell penetrating peptide scaffold (ACPP-AZD7762). In contrast to receptor directed ADCs, ACPPs redirect extracellular tumor microenvironment proteases to guide drug delivery. Intact, ACPPs are charge neutral which blocks the conjugated drug from cell entry. Upon cleavage by tumor overexpressed MMPs, the ACPP scaffold releases drug conjugated polycationic peptide which is then internalized by cells. We found that while cetuximab-AZD7762 ADC had high affinity for EGFR overexpressing tumor cells and prolonged blood circulation in murine models, cetuximab-AZD7762 lacked anti-tumor efficacy. On the other extreme, naked r9-AZD7762 drug conjugate was indiscriminately cytotoxic and radiosensitized a panel of tumor cell lines in cell culture: CAL27, A549, PANC1 and HCT116. However, r9 cell penetrating peptides lacked tumor specificity when intravenously injected into mice. In contrast, MMP-2/9 sensitive ACPPs balanced tumor selectivity with drug efficacy. Intact, ACPPs held conjugated AZD7762 in a pro-drug inactive state that upon tumor microenvironment ACPP cleavage resulted in tumor selective drug accumulation and anti-tumor efficacy in combination with IR. Importantly, a control non-cleavable ACPP-AZD7762 did not improve tumor control with IR, demonstrating the necessity of ACPP cleavage to release active drug. These results highlight the utility of the ACPP technology platform to target amine containing radiosensitizing drugs to tumor tissues based on extracellular protease activity that overcome the limitations of ADC conjugated radiosensitizers. Citation Format: Dina V. Hingorani, Maria F. Camargo, Maryam A. Quraishi, Joseph Aguilera, Stephen R. Adams, Sunil J. Advani. Redirecting tumor extracellular proteases to target radiosensitizer delivery [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6272.

Autophagy Induced by Proteasomal DUB Inhibitor NiPT Restricts NiPT-Mediated Cancer Cell Death

Ubiquitin–proteasome system (UPS) and autophagy–lysosome pathway (ALP) are two major systems for protein quality control (PQC) in eukaryotic cells. Interconnectivity between these two pathways has been suggested, but the molecular detail of how they impact each other remains elusive. Proteasomal deubiquitinase (DUB) is an important constituent in the UPS and has proved to be a novel anticancer target. We have previously found that a novel DUB inhibitor, nickel complex NiPT, induces apoptosis in both cultured tumor cell lines and cancer cells from acute myeloid leukemia human patients. In this study, we found that NiPT triggered autophagy both in vitro and in vivo. Mechanistically, NiPT targets two DUBs, USP14, and UCHL5, and increased the total cellular level of polyubiquitination. Deletion of the Ubiquitin Associated (UBA) domain of P62 that is required for polyubiquitin binding prevented NiPT-induced autophagy. NiPT-induced autophagy is through either concomitant activation of AMP-activated protein kinase (AMPK) and inhibition of mechanistic target of rapamycin (mTOR) signaling, or eliciting endoplasmic reticulum (ER)-stress by activating activating transcription factor 4 (ATF4) and C/EBP-homologous protein (CHOP). Moreover, NiPT could induce more lung cancer cells undergoing apoptosis if it synergistically uses autophagy inhibitors, suggesting that NiPT-induced autophagy protects cancer cell from death. Collectively, our findings demonstrate that autophagy inhibition enhances the anticancer effects of proteasomal DUB inhibitor and might be an effective treatment strategy for lung cancer.

Moving forward to address key unanswered questions on targeting PD-1/PD-L1 in cancer: limitations in preclinical models and the need to incorporate human modifying factors

The tremendous clinical success of immune checkpoint inhibition (ICI), particularly targeting the programmed cell death protein 1 (PD-1)/programmed death-ligand 1/2 (PD-L1/2) pathway, has resulted in application to multiple cancers, as a monotherapy and as a companion to both conventional and novel agents. Despite this, the precise mechanisms underlying the anti-tumor effects of PD-1/PD-L1 blockade remain unclear. Emphasis has centered on its reversal of tumor-specific CD8+ T-cell exhaustion, although many cell types and processes are likely impacted. Due to the complex and pervasive roles of PD-1/PD-L1 on T-cell biology, including on initial T-cell priming, PD-1 blockade likely affects all aspects of T- cell responses, and these other effects may be even more critical for durable anti-tumor responses. Delineating these complex interactions necessitates in vivo modeling. By far, the healthy, young and inbred laboratory mouse, transplanted with an extensively cultured tumor cell line, has been the predominant preclinical model used to assess potential therapeutic efficacies. However, these mouse models often do not adequately reflect the tumor progression and cellular and genetic heterogeneity found within human cancers. Furthermore, laboratory mice also present with a vastly restricted immune profile compared to humans. This commentary discusses some of the critical questions that need to be addressed to optimize the use of ICI as well as caveats and limitations for consideration when extrapolating preclinical mouse data to the human cancer scenario.

Two novel anthraquinones with cytotoxicity from Hedyotis caudatifolia

Seven anthraquinones, including two new natural products, hedyanthraquinones A and B (1, 2), were isolated from Hedyotis caudatifolia. The structures were elucidated based on NMR, MS data and physico-chemical properties. Compounds 1 and 2 displayed moderate cytotoxic activities against a panel of cultured tumor cell lines (HL-60, Bcap37, SMMC7721) with IC50 values of 13.71 ∼ 26.29 μM. The result showed that anthraquinones are typical compounds of Hedyotis genus, which could be useful as characteristic markers in chemotaxonomic research and might helpful for explaining the use of H. caudatifolia in Traditional Chinese Medicine.

Aporphine Alkaloids from Illigera aromatica from Guangxi Province, China

Three undescribed aporphine alkaloids laurodionine B (1), illigerine A (2), and N-formyl-laurolitsine (3) were isolated from the methanolic extracts of the Chinese medicinal plant, Illigera aromatica, together with three known analogues (4–6). The chemical structures of 1–6 were identified by spectroscopic methods including 1D and 2D NMR (1H, 13C, COSY, HSQC, and HMBC) and high resolution mass spectrometry (HRESIMS). Compounds 1–3 showed moderate inhibitory activities in vitro against two cultured tumor cell lines, Hela and SMMC7721, with IC50 values of 32.42–62.90 μM. Only compound 1 had in vitro cytotoxic activity against Bcap37 cells, with the IC50 value of 90.61 μM.

Abstract 2757: Antibody-drug conjugate payloads induce markers of immunogenic cell death in cancer cells

Abstract Activating the immunologic destruction of cancer cells is a clinically validated therapeutic approach. Immunogenic cell death (ICD) is a reported mechanism of activating the immune system wherein anti-cancer treatments induce specific tumor cell markers, followed by dendritic cell activation and T-cell recruitment. Antibody-drug conjugates (ADCs) are targeted biotherapeutics which deliver cytotoxins to cancer cells. Our hypothesis is that ADCs also may have immune-modulating properties and that the payloads delivered by ADCs may induce ICD. We developed three assays to evaluate ICD in cultured tumor cell lines: (1) HMGB1 extracellular protein levels, (2) cell surface calreticulin translocation, (3) and ICD-induced dendritic cell activation (ICD-DCA). First, cytototoxity was assessed for all compounds, then ICD studies were conducted at dose-multiples relative to each compound's anti-proliferative IC50. Next, cells were treated with various chemotherapeutic agents and extracellular HMGB1 protein detected by ELISA. However, HMGB1 levels strongly correlated with cytotoxicity, and did not differ among treatments. We next assessed calreticulin (CRT), an ER-resident protein reportedly translocated to the cell surface upon ICD. We evaluated the specificity of commercial antibodies to detect CRT in human (A549) or murine (EMT6) cell lines. Cells were incubated with CRT siRNA specific for murine or human sequences. Flow cytometry (for cell surface CRT) and immunoblots (for total CRT) confirmed that a commonly used CRT antibody detects CRT in murine cells, but not human cells. Murine EMT6, 4T1, B16F10, or CT26 cells were then treated with compounds of varied mechanisms-of-action. Our assays confirm previous reports that anthracycline chemotherapeutics (e.g., doxorubicin) induce CRT surface translocation. We also discovered that analogs of both the calicheamicin and cyclopropylpyrroloindolone (CPI) DNA inhibitor class of ADC payloads robustly induced CRT surface translocation in several murine cancer lines within 24 hr, even at relatively low subtoxic doses. Some DNA inhibitors did not induce CRT. Microtubule inhibitor auristatins and mertansine (DM1) induced CRT translocation, but at high cytotoxic doses and after 48 hr incubation. Next, tumor cells were treated with compounds, washed, then co-cultured with murine bone-marrow derived dendritic cells (BMDC). The DNA inhibitors which induced CRT on tumor cells also increased levels of CD86 activation marker and cytokine release from BMDC in these co-cultures. Hence, we have demonstrated that calreticulin, a reported marker of ICD, is activated in cultured murine tumor cells by unconjugated payloads typically delivered by ADCs. These compounds can also induce DC activation in tumor:BMDC co-cultures. These data suggest the potential for immune system activation using cytotoxic ADC payloads. Citation Format: Xingzhi Tan, My-Hanh Lam, Shoba Ragunathan, Keziban Unsal-Kacmaz, Frank Loganzo. Antibody-drug conjugate payloads induce markers of immunogenic cell death in cancer cells [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 2757.

PDLIM7 and CDH18 regulate the turnover of MDM2 during CDK4/6 inhibitor therapy-induced senescence

CDK4/6 inhibitors are being used to treat a variety of human malignancies. In well-differentiated and dedifferentiated liposarcoma their clinical promise is associated with their ability to downregulate the MDM2 protein. The downregulation of MDM2 following treatment with CDK4/6 inhibitors also induces many cultured tumor cell lines derived from different types of malignancies to progress from quiescence into senescence. Here we used cultured human cell lines and defined a role for PDLIM7 and CDH18, regulating MDM2 protein in CDK4/6 inhibitor-treated cells. Materials from our previous phase II trials with palbociclib were then used to demonstrate that expression of CDH18 protein was associated with response, measured as both progression-free survival and overall survival. This supports the hypothesis that the biologic transition from quiescence to senescence has clinical relevance for this class of drugs.

Unusual 28, 29-nor-9, 19 cycloartane triterpenoids from Chinese medical plant Streptocaulon griffithii Hook

Phytochemical investigation of the root of Streptocaulon griffithii Hook (Asclepiadaceae) led to the isolation of three unusual novel triterpenoids, 28, 29-nor-3β, 4β-dihydroxyl-9, 19-cycloartan-26-acid (1), 28, 29-nor-3β, 4β-dihydroxyl-9, 19-cycloartan- 26-acid methylester (2), a 30-nor-lupeol derivative 30-nor-3β-acetoxy-lupan-20-one (3) and five known compounds 4–8. Their structures were established on the basis of physical and spectroscopic analysis, including 1D and 2D NMR (1H, 13C, COSY, HSQC, and HMBC), ESI mass spectrometry, and by comparison with the NMR data published in the literature. The cytotoxicities of the isolated compounds against a panel of cultured tumor cell lines (Hela, PC3, SMMC7721, CNE) were evaluated. The new compounds 1 and 2 showed moderate activities with IC50 values of 11.76∼26.52μg/mL, respectively. The result showed that triterpenoids are typical compounds of Streptocaulon genus, which could be useful as characteristic markers in chemotaxonomic research and might helpful for explaining the use of S. griffithii in traditional medicine.

Structural Characterization of Cholestane Rhamnosides from Ornithogalum saundersiae Bulbs and Their Cytotoxic Activity against Cultured Tumor Cells.

Previous phytochemical studies of the bulbs of Ornithogalum saundersiae, an ornamental perennial plant native to South Africa, resulted in the isolation of 29 new cholestane glycosides, some of which were structurally unique and showed potent cytotoxic activity against cultured tumor cell lines. Therefore, we aimed to perform further phytochemical examinations of methanolic extracts obtained from Ornithogalum saundersiae bulbs, isolating 12 new cholestane rhamnosides (1–12) and seven known compounds (13–19). The structures of the new compounds (1–12) were identified via NMR-based structural characterization methods, and through a sequence of chemical transformations followed by spectroscopic and chromatographic analysis. The cytotoxic activity of the isolated compounds (1–19) and the derivatives (1a and 6a) against HL-60 human promyelocytic leukemia cells and A549 human lung adenocarcinoma cells was evaluated. Compounds 10–12, 16, and 17 showed cytotoxicity against both HL-60 and A549 cells. Compound 11 showed potent cytotoxicity with an IC50 value of 0.16 µM against HL-60 cells and induced apoptotic cell death via a mitochondrion-independent pathway.

Lithium protects hippocampal progenitors, cognitive performance and hypothalamus-pituitary function after irradiation to the juvenile rat brain.

Cranial radiotherapy in children typically causes delayed and progressive cognitive dysfunction and there is no effective preventive strategy for radiation-induced cognitive impairments. Here we show that lithium treatment reduced irradiation-induced progenitor cell death in the subgranular zone of the hippocampus, and subsequently ameliorated irradiation-reduced neurogenesis and astrogenesis in the juvenile rat brain. Irradiation-induced memory impairment, motor hyperactivity and anxiety-like behaviour were normalized by lithium treatment. Late-onset irradiation-induced hypopituitarism was prevented by lithium treatment. Additionally, lithium appeared relatively toxic to multiple cultured tumour cell lines, and did not improve viability of radiated DAOY cells in vitro. In summary, our findings demonstrate that lithium can be safely administered to prevent both short- and long-term injury to the juvenile brain caused by ionizing radiation.