The Optimization of HL60-IL6 Assay and its Application in the Pyrogen Detection of Monoclonal Antibody

Background: The HL60-IL6 assay has been initially established, but the process of the assay and calculation was not simplified. And there are no reports on whether it can be applied to detect pyrogen contamination in the monoclonal antibody. Objective: The study aimed to improve the HL60/IL-6 assay and detect the pyrogens in the monoclonal antibody drug by HL60-IL6 assay. Methods: The human promyelocytic leukemia cell line (HL-60) was incubated with pyrogen standard solution, such as lipopolysaccharide (LPS), zymosan and lipoteichoic acid (LTA),or monoclonal antibody sample solution for 48 hours, and then cytokines interleukin-6 (IL-6),secreted from HL-60, were measured by ELISA. The study further described the standard curves on OD (Optical Density) value of IL-6 responding to pyrogen stimulation, and determined the content of pyrogen in the monoclonal antibody production after validation. In addition, the sensitivity of HL60 to three pyrogens was evaluated to establish one standard curve to determine endotoxin and non-endotoxin level. Then, the credibility of standard curves was evaluated. After improvement of the assay, 9 monoclonal antibody batches were assayed for pyrogens in parallel with the Rabbit Pyrogen Test (RPT) and HL60/IL-6 assay. Results: It was achieved that the standard curve between OD value of IL-6 and pyrogen concentration was established. Then, it was found that the sensitivity of HL60 responding to LPS was the weakest, as a result of which, only LPS standard curve needs to be described in each test for detection of pyrogens. Besides, to evaluate the credibility of standard curve, the parameters of the standard curve were restricted and the resulting interpretation was also specified. 3 Bevacizumab batches failed the RPT, which also showed pyrogenic contamination by the HL60/IL-6 assay. Conclusion: HL60-IL6 assay was improved and can be applied to pyrogen detection of monoclonal antibody.

N-Terpenyl Benzisoselenazolones—Evaluation of the Particular Structure-Bioactivity Relationship

Different activity of compounds with the same molecular formula but possessing alternative bonding arrangement or orientation of atoms in space is a very important issue when constructing molecules that can selectively interact with certain domains of the biological target. Herein, we present the synthesis of the new chiral benzisoselenazol-3(2H)-ones substituted on the nitrogen atom with three monoterpene moieties: p-menthane, pinane and carane. All derivatives were obtained by the reaction of an appropriate amine with 2-(chloroseleno)benzoyl chloride. The applied terpenyl amines were first synthetized by a multistep procedure starting from the corresponding alcohol (p-menthane system) or alkene (pinene and carene systems). Finally, all compounds were tested as antioxidants and antiproliferative agents on breast cancer MCF-7 and human promyelocytic leukemia cell line HL-60. The correlation between the structure of the obtained organoselenium compounds, representing examples of various isomers including enantiomers, diastereoisomers and regioisomers, and their bio-activity was thoroughly evaluated.

Bioselectivity Induced by Chirality of New Terpenyl Organoselenium Compounds

A series of new chiral benzisoselenazol-3(2H)-ones substituted on the nitrogen atom with three monoterpene moieties—p-menthane, pinane and carane—was synthesized. The compounds were obtained by the reaction of 2-(chloroseleno)benzoyl chloride with an appropriate terpene amine, first synthesized by a multistep methodology starting from the corresponding alcohol (p-menthane system) or alkene (pinene and carene systems). Compounds were tested as antioxidants and anticancer agents. The N-isopinocampheyl-1,2-benzisoselenazol-3(2H)-one was the best peroxide scavenger and antiproliferative agent on the human promyelocytic leukemia cell line HL-60. The N-menthyl-1,2-benzisoselenazol-3(2H)-one revealed the highest anticancer potential towards breast cancer line MCF-7. The influence of structure and chirality on the bio-activity of the obtained organoselenium compounds was thoroughly evaluated.

Apoptotic Effect of Boron Derivatives on HL-60 Acute Promyelocytic Leukemia Cell Line

Acute myeloid leukemia (AML) is the most common form of acute leukemia and the overall 5-year survival in AML patients is 40–45% in young patients and less than 10% in elderly patients. Therefore, there is still a need to improve therapeutic approaches. After development of boron-based anticancer drugs (such as Bortezomib), boron compounds have gained attention with possible anti-leukemic feature. In this study, we evaluated anti-tumoral effects of borax pentahydrate (BP) and disodium pentaborate decahydrate (DPD) on leukemic cell line (HL-60). Cell viability was assessed by MTT assay and apoptotic effect of the compounds were evaluated by flow cytometry and cleaved PARP level detection by western blot. We observed that BP (24 mM) and DPD (6 mM) exhibit anti-leukemic effect via induction of apoptosis. Our study suggest that BP and DPD have potential anti-leukemic effect, which needs to be confirmed by further wide-spectrum studies.

CD34+ Derived Microparticles Regulate Apoptosis in Normal and Acute Myeloid Leukemia Cells

Introduction Microparticles (MPs) are small vesicles 100nm-1μm derived from the apoptotic or stimulated cells. The mechanism of their production is distinctive from exosomes or apoptotic bodies. MPs have been detected in the blood in many pathological conditions associated mainly with endothelial injury, thrombosis and inflammation. Our previous study showed that MPs originated also from CD34+ cells of umbilical cord blood, which is an alternative source for hemopoeitic stem cell transplantation. MPs considered as markers of cell activation, as well as apoptosis. Apoptosis is a complex interaction network regulated either through death receptor like FAS or through the internal pathway of Bcl2, Bax. The two pathways activate the executor of cell death program, caspases. The aim of this study is to elucidate the role of MPs in apoptosis of hemopoietic cells. Methods Umbilical cord blood units (UCB) were collected after informed consent. The units that were used in this study, were rejected as not appropriate for transplantation due to low volume. The HL60 promyelocytic leukemia cell line was cultured in RPMI (Life Technologies) supplemented with 10% fetal calf serum (FCS) and 1% penicillin-strepromycin. CD34+ MPs were isolated from the plasma of UCBs after centrifugation and magnetic bead MACS purification (Miltenyi Biotec). The number of CD34+ MPs was estimated by flow cytometry using CD34-PE and Annexin V-FITC Abs. Mononuclear cells (MNC) were collected after density gradient centrifugation on lymphoprep (Fresenius) and were cultured for 3 and 6 days in the presence of CD34+ MPs. Viability assays were performed using 7-AAD in flow cytometry. In another set of experiments different numbers of CD34+ MPs were used in MNC cultures. RNA was extracted from MNC using Qiagen RNA extraction kit and reverse transcribed into cDNA using Superscript II reverse transcriptase (Invitrogen) with random primers (Promega). RT-PCR was performed using Platinum Pfx polymerase (Invitrogen). The primers for FAS, BCL2, BAX, caspase 3, survivin and GAPDH genes were used. The PCR products were analysed in an agarose gel electrophoresis. Results Cell viability increased in UCB derived MNC (UCB-MNC) incubated with CD34+ MPs (800 /ml) after 3 day vs. 6 days of culture. The UCB-MNC viability was higher using 800/ml CD34+ MPs vs. 400/ml. In contrast, CD34+ MPs (800 /ml vs. 400/ml) did not affect the viability in one day MNC culture. Purified CD34+ MPs were applied to UCB-MNC cultures and by RT-PCR was shown increased expression of BCL2 gene as well as FAS and caspase-3 genes. The promyelocytc cell line HL60 has been used in order to analyze the effect of CD34+ MPs in leukemic cells. The expression of Bcl2 was decreased in HL60 cells co-incubated with CD34+ MPs. This result shows an opposite effect of CD34+ MPs in the apoptotic gene Bcl2 for the HL60 cells indicating that there are different mechanisms of MP function in various cell types. Conclusions In this study we have identified and monitored the time- and dose-dependent effect of CD34-derived microparticles in the viability of UCB mononuclear cells. Additionally, CD34+ MPs function is accosiated with the high expression of the pro- and anti-apoptotic Bcl2 and apoptotic FAS. In contrast CD34+ MPs decreases the expression of Bcl2 in the promyelocytic leukemia cell line HL60. Therefore the stem cell derived microparticles might serve as a potential regulator of apoptosis in normal and malignant hematopoietic cells. Disclosures No relevant conflicts of interest to declare.