Leukemia Cell Line K562 Research Articles

Selectively and Efficiently Expanding Natural Killer Cells in Vitro

Natural killer (NK) cells are the main innate immune cells, and have powerful antitumor activity in vitro, but NK cells based immunotherapy does not often get satisfactory results in clinical practice due to insufficient number and poor activity of NK cells.. Therefore, how to improve expansion efficiency and killing activity of NK cells in vitro is critical. Here we report an in vitro culture system culturing peripheral blood mononuclear cells (PBMC) with irradiated LCL as feeder cells in 10%FBS/RPMI1640 culture medium in the presence of 100U/ml IL-2 and 5ng/ml IL-15 for 14 days could selectively expand NK cells. With this culture system, the purity of NK cells increased from 11.5%± 7.01% to 75.3±10.9% (P< 0.01), and the expansion efficiency was about 625±63 times than the original PBMC. Compared with the fresh NK cells isolated from PBMC, there was no significant change in the expression of inhibitory receptors on the surface of expanded NK cells, while the expression of activating receptors was significantly increased (p<0.05). The cytotoxicity of expanded NK cells against leukemia cell line K562 was significantly higher than that of freshly isolated NK cells (72± 5.6% vs 8.7±2.3%, E: T=1, P<0.01), expanded NK cells also showed stronger cytotoxicity to LCL (22±3.6%, E: T=1) and primary leukemia cells (42±5.2%, E:T=1). In summary, we could selectively expand NK cells with this in vitro culture system, the expanded NK cells gained higher expression of NCRs and NKG2D receptors, and displayed greater cytotoxicity against tumor cells, therefore could be readily applied to anti-tumor treatment in clinical treatment. Figure Disclosures No relevant conflicts of interest to declare.

Divergent Synthesis of Bioactive Dithiodiketopiperazine Natural Products Based on a Double C(sp3 )-H Activation Strategy.

This article provides a detailed report of our efforts to synthesize the dithiodiketopiperazine (DTP) natural products (-)-epicoccin G and (-)-rostratin A using a double C(sp3 )-H activation strategy. The strategy's viability was first established on a model system lacking the C8/C8' alcohols. Then, an efficient stereoselective route including an organocatalytic epoxidation was secured to access a key bis-triflate substrate. This bis-triflate served as the functional handles for the key transformation of the synthesis: a double C(sp3 )-H activation. The successful double activation opened access to a common intermediate for both natural products in high overall yield and on a multigram scale. After several unsuccessful attempts, this intermediate was efficiently converted to (-)-epicoccin G and to the more challenging (-)-rostratin A via suitable oxidation/reduction and protecting group sequences, and via a final sulfuration that occurred in good yield and high diastereoselectivity. These efforts culminated in the synthesis of (-)-epicoccin G and (-)-rostratin A in high overall yields (19.6 % over 14 steps and 12.7 % over 17 steps, respectively), with the latter being obtained on a 500 mg scale. Toxicity assessments of these natural products and several analogues (including the newly synthesized epicoccin K) in the leukemia cell line K562 confirmed the importance of the disulfide bridge for activity and identified dianhydrorostratin A as a 20x more potent analogue.

Anti-leukaemic and anti-melanoma activities of homoisoflavonoids from Pseudoprospero firmifolium subsp. natalensis (Hyacinthaceae)

Five homoisoflavonoids, (3S)-3,5-dihydroxy-7‑methoxy‑3-(3′,4′-dimethoxybenzyl)-4-chromanone, 1, (3S)-3,5-dihydroxy-7,8-dimethoxy-3-(3′‑hydroxy‑4′‑methoxy)-4-chromanone, 2, (3R)-7,8-dimethoxy-5‑hydroxy‑3-(4′-hydroxybenzyl)-4-chromanone, 3, and (3R)-5‑hydroxy‑7‑methoxy‑3-(3′,4′-dimethoxybenzyl)-4-chromanone, 4, (3S)-3,5,7-trihydroxy-3-(3′‑hydroxy‑4′-methoxybenzyl)-4-chromanone, 5, and a spirocyclic nortriterpenoid, 29‑hydroxy‑15-deoxyeucosterol, 6, were isolated from the bulbs of Pseudoprospero firmifolium subsp. natalensis. Compounds 1, 2 and 5 were isolated previously from P. firmifolium subsp. firmifolium although configurations at C-3 were not established previously. Configurations at C-3 have been confirmed in this work for compounds 1–4 using ECD spectroscopy. The current findings for Pseudoprospero, and most especially of compound 3, indicate that the capacity of the Hyacinthoideae to synthesize homoisoflavonoids evolved prior to the divergence of the three extant tribes (Hyacintheae, Massonieae and Pseudoprospereae) ca. 18.8 Ma ago. Compound 5 displayed activity against two leukemia cell lines SR (GI50 = 0.64 μM) and K-562 (GI50 = 0.86 μM), and the melanoma MDA-MB-435 cell line (GI50=0.41 μM).

WT1 activates transcription of the splice factor kinase SRPK1 gene in PC3 and K562 cancer cells in the absence of corepressor BASP1

Dysregulated alternative splicing plays a prominent role in all hallmarks of cancer. The splice factor kinase SRPK1 drives the activity of oncogenic splice factors such as SRSF1. SRSF1 in turn promotes the expression of splice isoforms that favour tumour growth, including proangiogenic VEGF. Knockdown (with siRNA) or chemical inhibition (using SPHINX) of SRPK1 in K562 leukemia and PC3 prostate cancer cell lines reduced cell proliferation, invasion and migration. In glomerular podocytes, the Wilms tumour suppressor zinc-finger transcription factor WT1 represses SRPK1 transcription. Here we show that in cancer cells WT1 activates SRPK1 transcription, unless a canonical WT1 binding site adjacent to the transcription start site is mutated. The ability of WT1 to activate SRPK1 transcription was reversed by the transcriptional corepressor BASP1, and both WT1 and BASP1 co-precipitated with the SRPK1 promoter. BASP1 significantly increased the expression of the antiangiogenic VEGF165b splice isoform. We propose that by upregulating SRPK1 transcription WT1 can direct an alternative splicing landscape that facilitates tumour growth.

Effects of Dasatinib on the Expansion, Subsets, Receptor Expression and Cytotoxic Function of NK Cells in Vitro

To investigate the effect of dasatinib on the expansion of NK cells in vitro, as well as the subsets, receptor expression and cytotoxic function of NK cells. Peripheral blood mononuclear cells (PBMCs) were isolated from healthy adult volunteers and cultured with SCGM added IL-2 and IL-15 for expansion of NK cells. In this culture system, dasatinib of different concentrations were added. Cell counting and phenotyping by flow cytometry were used to evaluate the amplification efficiency of NK cells. FCM was used to detect the expression of receptors on the surface of NK cells and the distribution of subsets. Subsequently, degranulation assay and CFSE/7AAD based cytotoxicity assay were used to detect the effects of dasatinib on NK cytotoxicity against leukemia cell line K562 cells. The expansion efficiency of NK cells in vitro could be increased by dasatinib at the concentration range of 5-50 nmol/L, and the expansion efficiency of NK cells reached the peak at 20 nmol/L of dasatinib. The NK cytotoxicity against K562 cells in dasatinib cultured group at the concentration of 20 nmol/L was significantly higher than that in control group. For the cells cultured by disatinib in vitro, the MFI of CD226, NKP46 and NKG2D was up-regulated; the ratio of NKG2A+CD57- subset was down-regulated, while the ratio of NKG2A-CD57+ subset was up-regulated.The degranulation response of NKG2A-CD57+ NK cells to K562 cells was stronger than that of NKG2A+CD57- NK cells. The results shows that appropriate dose of dasatinib(20 nmol/L) can increases the amplification efficiency of NK cells, simultaneously up-regulates the expression of NK activating receptors and increases the NKG2A-CD57+ subset, which lead to the enhancement of NK cytotoxicty against leukemia cell lines.

Checkpoint kinase‑1 inhibition and etoposide exhibit a strong synergistic anticancer effect on chronic myeloid leukemia cell line K562 by impairing homologous recombination DNA damage repair.

Leukemia, a malignant hematological disease, has poor therapeutic outcomes due to chemotherapeutic resistance. Increasing evidence has confirmed that the elevated capacity for DNA damage repair in cancer cells is a major mechanism of acquired chemotherapeutic resistance. Thus, combining chemotherapy with inhibitors of DNA damage repair pathways is potentially an ideal strategy for treating leukemia. Checkpoint kinase 1 (CHK1) is an important component of the DNA damage response (DDR) and is involved in the G2/M DNA damage checkpoint. In the present study, we demonstrated that shRNA-mediated CHK1 silencing suppressed cell proliferation and enhanced the cytotoxic effects of etoposide (VP16) in the chronic myeloid leukemia (CML) cell line K562 through the results of CCK-8, and comet assay. The results demonstrated that shRNA-induced CHK1 silencing can override G2/M arrest and impair homologous recombination (HR) repair by reducing breast cancer susceptibility gene 1 (BRCA1) expression. Cells had no time, and thus limited ability, to repair the damage and were thus more sensitive to chemotherapy after CHK1 downregulation. Second, we tested the therapeutic effect of VP16 combined with CCT245737, an orally bioavailable CHK1 inhibitor, and observed strong synergistic anticancer effects in K562 cells. Moreover, we discovered that CCT245737 significantly prevented the G2/M arrest caused by acute exposure to VP16. Interestingly, CCT245737 inhibited both BRCA1 and Rad51, the most important component of the HR repair pathway. In conclusion, these results revealed that CHK1 is potentially an ideal therapeutic target for the treatment of CML and that CCT245737 should be considered a candidate drug.

Identification of biomarkers associated with extracellular vesicles based on an integrative pan-cancer bioinformatics analysis.

Extracellular vesicle (EV) has received increasing attention over the last decade. However, biomarkers and mechanisms underlying remain largely limited. Three microarray profiles, GSE78718 (K562 leukemia cell line), GSE45301 (U87-MG glioblastoma cell line), and GSE9589 (SW480 colon cancer cell line), were analyzed for the overlapped differentially expressed genes (DEGs). SurvExpress was used for the prognostic analysis of hub genes signature. Predicted transcription factors networks were built by NetworkAnalysis. Characterization between hub genes and immune cells was analyzed by the tumor immune estimation resources (TIMER) and single-sample gene set enrichment analysis (ssGSEA). The most significantly enriched pathway was lysosome. Hub genes included lysosomal-associated membrane protein 1 (LAMP1), heat shock protein family A (Hsp70) member 5 (HSPA5), lysosomal-associated membrane protein 2 (LAMP2), integrin subunit alpha V (ITGAV), and transmembrane protein 30A (TMEM30A). Significant prognostic values of hub genes signature were identified in glioblastoma (P-value = 0.006), but not colon cancer. In colon cancer, ITGAV displayed remarkably high correlation with tumor immune infiltrating cells. In glioblastoma, the highest correlation was found between HSPA5 and dendritic cell. Moreover, distinct association of immune cells between cell and EV were identified via ssGSEA. This study identified biomarkers in EV with potential immunological insights and clinical values.

Inhibitory Effect of S1PR2 Antagonist JTE-013 on Proliferation of Chronic Myeloid Leukemia Cells

To investigate the effect of sphingosine-1-phosphate receptor 2 (S1PR2) specific antagonist JTE-013 on the proliferation of human chronic myeloid leukemia (CML) cell line K562. K562 cells were treated with JTE-013 (0, 0.5, 1, 5, 10, 20 μmol/L) for 24 and 48 hours respectively, CCK8 assay was used to detect the cell viability. K562 cells were treated with JTE-013 (0, 5, 10, 20 μmol/L) for 24 hours, propidium iodide (PI) DNA staining was used to analyze the cell cycle, Western blot was used to determine the levels of P21 and Cyclin D1 protein expression. JTE-013 inhibited the proliferation of CML cell line K562 in a dose dependent manner (r=-0.971). The proliferation rate of CML cells showed that the activity of CML cells decreased gradually with the increase of JTE-013 concentration (r=-0.971). The detection demonstrated that JTE-013 suppressed tumor cell proliferation through cell cycle arrest in G0/G1 phase. Further detection of the protein expressions of G1 phase regulators showed that level of P21 increased, and expression of Cyclin D1 decreased. JTE-013, a S1PR2 antagonist, can inhibit the proliferation of human CML K562 cells, which may be achieved by arresting the cells in G0/G1 phase.

Novel N-4-Piperazinyl Ciprofloxacin-Ester Hybrids: Synthesis, Biological Evaluation, and Molecular Docking Studies

A series of novel N-4-piperazinyl ciprofloxacin-ester hybrids has been synthesized and the structures confirmed by1H and 13C NMR, FT-IR spectral data, and elemental analysis. The products have been tested in vitro for their antibacterial activity against six bacterial strains (MRSA, Staphylococcus epidermidis, Bacillus subtilis, Escherichia coli,Salmonella enterica, and Klebsiella pneumoniae) and have demonstrated good antibacterial activity with MIC values range 6.25–200 µg/mL. Antifungal and cytotoxic activities of the products have been tested against Candida kefyr and human leukemia K562 cell line, respectively. All compounds inhibit growth of K562 cells more efficiently than the parent ciprofoxacin in a dose- and duration-dependent way. Molecular docking studies performed for the compound 3i indicates that similarly to ciprofloxacin it can act as an inhibitor of S. aureus DNA gyrase.

Honokiol-Chlorambucil Co-Prodrugs Selectively Enhance the Killing Effect through STAT3 Binding on Lymphocytic Leukemia Cells In Vitro and In Vivo

The broad-spectrum DNA alkylating therapeutic, chlorambucil (CBL), has limited safety and shows lower therapy effect because of a short half-life while used in the clinic. Therefore, it is very necessary to develop a more efficient and safer type of CBL derivate against tumors with selective targeting of cancer cells. In addition, the natural product of honokiol (HN), the novel potent chemo-preventive or therapeutic entity/carrier, can target the mitochondria of cancer cells through STAT3 to prevent cancer from spreading and metastasizing. In this study, we designed and synthesized the honokiol–chlorambucil (HN–CBL) co-prodrugs through carbonate ester linkage conjugating with the targeted delivery help of the HN skeleton in cancer cells. Biological evaluation indicated that HN–CBL can remarkably enhance the antiproliferation of human leukemic cell lines CCRF-CEM, Jurkat, U937, MV4-11, and K562. Furthermore, HN–CBL can also selectively inhibit the lymphocytic leukemia (LL) cell survival compared to those mononuclear cells derived from healthy donors (PBMCs), enhance mitochondrial activity in leukemia cells, and induce LL cell apoptosis. Molecular docking and western blot study showed that HN–CBL can also bind with the STAT3 protein at some hydrophobic residues and downregulate the phosphorylation level of STAT3-like HN. Significantly, HN–CBL could dramatically delay leukemia growth in vivo with no observable physiological toxicity. Thus, HN–CBL may provide a novel and effective targeting therapeutic against LL with fewer side effects.

Targeting the MAPK/ERK and PI3K/AKT Signaling Pathways Affects NRF2, Trx and GSH Antioxidant Systems in Leukemia Cells.

The mitogen-activated protein kinase (MAPK)/extracellular signal kinase (ERK) and phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signal transduction pathways have been implicated in the pathogenesis of leukemia. The aim of this study was to investigate the effect of the combination of ERK1/2 inhibitor AZD0364 and PI3K inhibitor ZSTK474 on acute lymphoblastic leukemia (ALL) REH, MOLT-4, acute myeloid leukemia (AML) MOLM-14, and chronic myeloid leukemia (CML) K562 cell lines. To evaluate the interactions of the drugs, cells were treated for 48 h with AZD0364 or ZSTK474 alone and in combination at fixed ratios. The combinatorial effects of both inhibitors were synergistic over a wide range of concentrations in REH, MOLT-4, and MOLM-14 cell lines. However, in K562 cells, the effects were found to be antagonistic. Furthermore, AZD0364 and ZSTK474 significantly decreased both ERK1/2 and AKT activation in REH, MOLT-4, and MOLM-14 cells. The results showed that incubation with both AZD0364 and ZSTK474 inhibited cell viability, increased reactive oxygen species (ROS) production, and induced apoptosis in leukemia cells. We observed that combined treatment with AZD0364 and ZSTK474 affected nuclear factor-κB (NF-κB) and antioxidant protein levels: NF-E2-related factor 2 (NRF2), heme oxygenase-1 (HO-1), thioredoxin (Trx), thioredoxin reductase (TrxR), and the reduced glutathione/oxidized glutathione (GSH/GSSG) ratio. These effects were accompanied with decreased antiapoptotic survivin protein level. However, distinct cell line dependent effects were observed. In conclusion, the combination of AZD0364 and ZSTK474 can exert a synergistic anticancer effect in ALL and AML cells, which is associated with the induction of oxidative stress and the involvement of cellular antioxidant defense mechanisms.

Transport of ribavirin in human myelogenous leukemia cell line K562.

The anticancer effect of ribavirin, a purine nucleoside analogue, has been studied using cultured cancer cells such as the human myelogenous leukemia cell line K562. In order to exert its pharmacological effect, ribavirin has to enter cancer cells. However, there is little information concerning the transport mechanism of ribavirin into K562 cells. In this study, therefore, we examined the uptake mechanism of ribavirin in K562 cells. The uptake of ribavirin in K562 cells was time- and temperature-dependent, and was saturable with a Km value of 1.5 mM. Ribavirin uptake was inhibited by nucleosides such as adenosine and uridine, and by inhibitors of equilibrative nucleoside transporter 1 (ENT1) such as S-(4-nitrobenzyl)-6-thioinosine and dipyridamole in a concentration-dependent manner. In addition, the expression of ENT1 mRNA in K562 cells was confirmed by real-time PCR. On the other hand, Na+-dependence of ribavirin uptake was not observed, suggesting the involvement of ENT1, but not Na+-dependent concentrative nucleoside transporters, in ribavirin uptake in K562 cells. Treatment of K562 cells with sodium butyrate induced erythroid differentiation, but ribavirin uptake activity and sensitivity of the uptake to various inhibitors were not different between native and differentiated K562 cells. These results suggest that ribavirin uptake into K562 cells is mainly mediated by ENT1, which may have a pivotal role in anticancer effect of ribavirin.

Cytotoxic Effects of Organotin(IV) Dithiocarbamate Compounds with Different Functional Groups on Leukemic Cell Line, K-562

Cancer is one of the main causes of mortality in the world. New compounds are currently being synthesized to overcome this disease. Organotin is getting more attention as anticancer agent due to its potent cytotoxic properties towards cancer cells. In this study, a series of newly synthesized organotin compounds known as dimethyltin(IV) (compound 1), dibutyltin(IV) (compound 2) and triphenyltin(IV)methylisoprophyldithiocarbamate (compound 3) have been assessed for their cytotoxic effect towards leukemic cell line K-562. MTT [3-(4,5-dimethylthiozol-2-yl)-2,5-diphenyltetrazolium bromide] assay was employed to determine the cytotoxic effect for each of the compounds at different time points and concentrations. Compound 2 and 3 exhibited potent cytotoxic effects towards K-562 cells with IC50 values less than 2 µM. Compound 3 was found as the most toxic towards K-562 cells based on the lowest IC50 value for 72 h period of treatment at 0.25 µM. Morphological observation was conducted with IC50 values at 24, 48, and 72 h. Results showed that the changes were first observed as early as 24 h and the most significant changes were observed after 72 h period of treatment with the characteristic of apoptosis such as cell shrinkage and membrane blebbing. In addition, necrosis characteristic such as cell lysis and swelling had also been observed. Organotin(IV) methylisopropyldithiocarbamate with different functional groups affect the cytotoxic properties towards leukemic cells K-562 and these compounds have potential to be developed as an anticancer agent through structural modification.

Olmutinib Reverses Doxorubicin Resistance in ETS1-Overexpressing Leukemia Cells.

BackgroundDrug resistance is a major problem in the treatment of leukemia with doxorubicin (Dox), and the erythroblastosis virus E26 oncogene homolog 1 (ETS1) gene is associated with drug resistance. Olmutinib is a third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) reported to play a role in reversing multidrug resistance (MDR) in cancer cells. The objective of this study was to investigate whether olmutinib could reverse Dox resistance in leukemia cells overexpressing ETS1.Material/MethodsHuman chronic myelogenous leukemia cell line K562 and its Dox-resistant cell line K562/ADR were used. Western blot and qPCR detected the expression of ETS1 and ABCB1. Cell proliferation was measured by cell counting kit-8 and methyl thiazolyl tetrazolium. Cell apoptosis was observed by western blot and flow cytometry. A nude mice K562/ADR xenograft model was used to investigate the inhibitory effects of olmutinib on tumor growth in vivo.ResultsThe mRNA and protein expressions of ETS1 and ABCB1 were up-regulated in Dox-resistant leukemia cell line K562/ADR. We overexpressed ETS1 in both cell lines, finding that olmutinib inhibited the cell viability of K562 and K562/ADR in a concentration-dependent manner. The cytotoxicity of Dox to EST1-overexpressing K562/ADR cells was enhanced by olmutinib. Olmutinib also promoted apoptosis of K562 and K562/ADR cells compared with Dox treatment alone. In vivo, olmutinib enhanced the inhibitory effects of Dox on ETS1-overexpressing K562/ADR cell xenograft growth.ConclusionsOur results suggest that the novel EGFR TKI olmutinib enhances the sensitivity of ETS1-overexpressing leukemia cells to Dox.

Cytotoxic Activity of Germacrane-Type Sesquiterpene Lactones from Dimerostemma aspilioides.

The need for effective candidates as cytotoxic drugs that at the same time challenge cancer multidrug resistance encouraged a search for these in plants of central Argentina. Bioassay-guided fractionation of the cytotoxic extract from Dimerostemma aspilioides led to the isolation of the germacranolide tomenphantin A (1), along with three new analogues (2-4). These efficiently inhibited the proliferation of the leukemia cell lines K562 and CCRF-CEM and their resistant variants, Lucena 1 and CEM/ADR5000, respectively, with IC50 values ranging from 0.40 to 7.7 μM. The structures and relative configurations of compounds 1-4 were elucidated by analysis of the spectroscopic data, in particular NMR spectroscopy. The most active among these was compound 1 (IC50 = 0.40-5.1 μM), and, therefore, this was selected as a model for a mechanistic study, which revealed that its antiproliferative effect was mediated by cell cycle arrest in the G2/M phase followed by apoptosis. The activity of compound 1 was selective, given the absence of cytotoxicity toward peripheral blood mononuclear cells. The results show the potential of these compounds, and in particular of compound 1, as leads for the development of drug candidates to fight sensitive and resistant leukemia cells.

Co-PRECIPITATION SYNTHESIS, CHARACTERIZATION OF CoFE2O4 NANOMATERIAL AND EVALUATION OF ITS TOXICITY BEHAVIOR ON HUMAN LEUKEMIA CANCER K562 CELL LINE

In this study, cobalt ferrite nanomaterial with inverse spinel structure was synthesized via a co-precipitation method using Co(NO3)3.6H2O and Fe(NO3)3.9H2O. A 15% NH4OH solution was used as an alkaline agent for pH adjustment. Oleic acid was used as a coating agent on the surface of the synthesized cobalt ferrite nanoparticles. PXRD analysis confirmed that the obtained material had a main CoFe2O4 cubic crystal structure with space group Fd-3m and lattice parameters a=b=c= 8.40 A (JCPDS standard card no = 01-077-0426). According to the SEM analysis, the pseudo-spherical particles size distribution and morphology of the as-synthesized nanomaterial were homogeneous. The magnetic behavior of the obtained material was analyzed by vibrating-sample magnetometer (VSM). Besides, anti-cancer effect of the as-synthesized cobalt ferrite nanoparticles was examined on an experimental model of acute myeloid leukemia (CML) K562 cell line for the first time. Inhibition of cell growth and cytotoxicity (apoptosis) that are characteristics features of cobalt ferrite nanoparticles are tested on K562 cell line. The results showed growth and viability inhibition and induction of apoptosis in K562 cells treated with the nanoparticles.

Anti-PD-1 responsive circulating immune cells as biomarkers of response to neoadjuvant chemotherapy in HER2+ breast cancer (BC).

e12646 Background: HER2+ BC patients (pts) achieving a pathological complete response (pCR) to neo-adjuvant therapy have better long-term survival outcomes. Alternative treatment strategies may be more beneficial for pts that will not achieve a pCR but there are no predictive biomarkers for stratification of pts to appropriate clinical studies. Antibody-dependent cell-mediated cytotoxicity (ADCC) mediated by immune cells like NK cells can be elicited by therapeutic antibody therapies including the HER2-targeting trastuzumab (T). We hypothesised that the presence of immune checkpoint-inhibited ADCC-mediating immune cells in the blood may identify pts with an immune-suppressed tumour microenvironment who will not achieve a pCR. We used an in vitro functional assay to assess the PD-1-inhibited ADCC response of pre-treatment peripheral blood mononuclear cells (PBMCs) from HER2+ BC pts as a potential biomarker of pCR. Methods: Pre-treatment PBMC samples (n = 21) were obtained from HER2+ BC pts who received neo-adjuvant chemotherapy (docetaxel, carboplatin) with a HER2-targeted therapy (T n = 5, T and lapatinib (L) n = 13, or L alone n = 3), ICORG 10-05 (NCT01485926). Pt response was determined as pCR (n = 7), partial response (PR, n = 10) or non-response (NR, n = 4). The immune function assay used a flow cytometry-based method to determine the T (10µg/µl)-mediated ADCC (T-ADCC) capacity of the PBMCs +/- the anti-PD-1 therapy pembrolizumab (P) (10µg/µl). The HER2+ BC cell line SKBR3 was used as the target cell line. Direct PBMC-mediated cytotoxicity against the non-MHC Class I-restricted leukemic K562 cell line was also determined. Statistical analysis was carried out using the MedCalc program. Results: There was no significant difference in direct cytotoxicity against K562 (pCR vs NR p = 0.13, pCR vs PR p = 0.52, PR vs NR p = 0.40), or in T-ADCC against SKBR3 (pCR vs NR p = 0.71, pCR vs PR p = 0.52, PR vs NR p = 0.32), between response cohorts in the absence of P. When T-ADCC was examined in individual pt samples, the addition of P resulted in significant increases in T-ADCC vs T-ADCC in the absence of P in 3/4 NR (p = 0.03, p = 0.01, p = 0.002) and 4/10 PR (p = 0.003, p = 0.007, p = 0.009, p = 0.01). No significant increase in T-ADCC was observed in the pCR cohort (0/7) as a result of the addition of P. Using pre-treatment PBMCs, the assay detected 7/14 (50%) of HER2+ BC pts who did not achieve a pCR. Conclusions: PD-1-inhibited ADCC-capable immune cells may identify a subset of HER2+ BC pts who will not achieve a pCR. Further investigation is warranted using a larger patient cohort.

A new cytotoxic isoflavane from Dalbergiae Odoriferae Lignum

Fourteen compounds were isolated from the ethanol extract of Dalbergiae Odoriferae Lignum by various chromatographic techniques, including column chromatographies on silica gel, Sephadex LH-20 and semi-preparative HPLC. Their structures were determined by spectroscopic techniques as S-3'-hydroxy-7,2',4'-trimethoxyisoxane(1), 2-(2',4'-dimethoxyphenyl)-6-hydroxybenzofuran(2), 2-(2'-hydroxy-4'-methoxyphenyl)-6-methoxybenzofuran(3), 7,2',4'-trimethoxydihydroisoflavone(4), sativanone(5), 3,9-dimethoxy-6H-benzofuro[3,2-c]chromen-6-one(6),(6 aS,11 aS)-homopterocarpin(7),(6 aS,11 aS)-8-hydroxy-3,9-dimethoxypterocarpan(8),(6 aS,11 aS)-3,8,9-trimethoxypterocarpan(9), isodalbergin(10), isoliquiritigenin(11), butein(12), butin(13) and 3,7,4'-trihydroxyflavone(14). Among them, compound 1 was a new compound, while 2 and 3 were new natural products, 6, 8, 9 and 14 were isolated for the first time from Dalbergiae Odoriferae Lignum. Compounds 1-14 were tested for their cytotoxic activity against human hepatoma cell line BEL-7402, human gastric cancer cell line SCG-7901, human lung cancer cell line A549, human chronic myeloid leukemia cell line K562 and HeLa human cervical cancer cellline by MTT method. Compound 1 exhibited significant cytotoxicity with IC_(50) values ranging from 2.85 to 11.62 μg·mL~(-1). In addition, 2, 11 and 12 showed weak cytotoxic activities.

Expansion of human γ9δ2 T cells to target hematologic malignancies

Background & Aim γδ T cells are multivalent immune cells that sense a broad spectrum of tumor-associated stress patterns by germline-encoded receptors to lyse them without prior potentiation. There is an unmet need in India to develop an affordable universal therapeutic immune cell product. The therapeutic expansion of the γ9δ2 T cell subset with zoledronic acid and IL-2 is being explored for clinical applications. Xenogeneic origin of the supplemented serum is a challenge in the cGMP level expansion of γδ T cells. Therefore, we aimed to optimize a cGMP compatible protocol for the expansion of γ9δ2 T cells in a serum-free media to be used for adoptive immunotherapy applications at CSCR/CMC. Methods, Results & Conclusion We compared the expansion and function of γ9δ2 T cells in serum-supplemented (RPMI+10%FBS) and serum-free (Optimizer®) media. Peripheral blood mononuclear cells (PBMC) from healthy donors were activated by 5µM Zoledronic Acid (ZA) with IL-2 (1000IU) for 14 days. Cell proliferation, fold expansion, and phenotype were monitored. The antitumor functions were assessed against K562 cell lines by a FACS based tumor-toxicity assay. Among the normal donors, the baseline levels of CD3+γ9+δ2+ T cells were 5.5±1.043% (mean±sem, range 0.2-20.9, n=15). There was a robust expansion of γ9δ2 T cells in the serum-supplemented media (258.26±55.84-fold, range 42.92- 472, n=8) compared to serum-free media (109.62±29.89-fold, range 42.95-296, n=8). IL-21 might help resolving the variations in the fold expansion. An abrupt reduction of αβ T cells was observed by day 7 with a concomitant increase in the CD3+γ9+ T cells during the course of the expansion. After two weeks, 87.82±5.11% (n=8) of T cells were CD3+γ9+ in Optimizer™ with more than 90% of the CD3+γ9+ T cells positive for δ2+. γ9δ2 T cells from the patients of multiple myeloma were also expanded with ZA and IL-2 albeit with reduced fold expansion (7.89 and 45.97, n=2) and δ2 percentage within CD3+γ9+ population (44.3 and 67%). In a 4hr FACS based cytotoxicity assay, the expanded γ9δ2 T cells directly lysed the leukemia cell lines K562 in an effector cell number dependent manner. In conclusion, we have optimized a cGMP compatible protocol to expand γ9δ2 T cells in a serum-free media to target multiple hematologic malignancies in the NSG animal models and eventually in the clinic.

Design, Synthesis of Novel Tetrandrine-14-l-Amino Acid and Tetrandrine-14-l-Amino Acid-Urea Derivatives as Potential Anti-Cancer Agents.

Tetrandrine, a dibenzyltetrahydroisoquinoline alkaloid isolated from the root of the traditional Chinese medicinal plant Stephania tetrandra S. Moore, a member of the Menispermaceae, showed anti-cancer activity by inhibiting cell proliferation, preventing cell cycle progress and induction of cell death and autophagy. In this study, twelve tetrandrine-l-amino acid derivatives and twelve tetrandrine-14-l-amino acid-urea derivatives were designed and synthesized, using C14-aminotetrandrine as raw material. Then the preliminary in vitro anti-cancer activities of these derivatives against human breast cancer cell line MDA-MB-231, human leukemia cell lines HEL and K562 were evaluated. The in vitro cytotoxicity results showed that these derivatives exhibited potent inhibitory effects on cancer cell growth, and the primary structure-activity relationships were evaluated. Notably, compound 3f exhibited satisfactory anticancer activity against all three cancer cell lines, especially the HEL cell line, with the IC50 value of 0.23 µM. Further research showed that 3f could induce G1/S cycle arrest and apoptosis in a dose- and time- dependent manner on the leukemia cell line HEL. The results suggested that 3f may be used as a potential anti-cancer agent for human leukemia.