Evaluation of Nonviral piggyBac and lentiviral Vector in Functions of CD19chimeric Antigen Receptor T Cells and Their Antitumor Activity for CD19+ Tumor Cells

Nonviral transposon piggyBac (PB) and lentiviral (LV) vectors have been used to deliver chimeric antigen receptor (CAR) to T cells. To understand the differences in the effects of PB and LV on CAR T-cell functions, a CAR targeting CD19 was cloned into PB and LV vectors, and the resulting pbCAR and lvCAR were delivered to T cells to generate CD19pbCAR and CD19lvCAR T cells. Both CD19CAR T-cell types were strongly cytotoxic and secreted high IFN-γ levels when incubated with Raji cells. TNF-α increased in CD19pbCAR T cells, whereas IL-10 increased in CD19lvCAR T cells. CD19pbCAR and CD19lvCAR T cells showed similar strong anti-tumor activity in Raji cell-induced mouse models, slightly reducing mouse weight while enhancing mouse survival. High, but not low or moderate, concentrations of CD19pbCAR T cells significantly inhibited Raji cell-induced tumor growth in vivo. These CD19pbCAR T cells were distributed mostly in mesenteric lymph nodes, bone marrow of the femur, spleen, kidneys, and lungs, specifically accumulating at CD19-rich sites and CD19-positive tumors, with CAR copy number being increased on day 7. These results indicate that pbCAR has its specific activities and functions in pbCAR T cells, making it a valuable tool for CAR T-cell immunotherapy.

Preparation and In Vitro Evaluation of RITUXfab-Decorated Lipoplexes to Improve Delivery of siRNA Targeting C1858T PTPN22 Variant in B Lymphocytes

Autoimmune endocrine disorders, such as type 1 diabetes (T1D) and thyroiditis, at present are treated with only hormone replacement therapy. This emphasizes the need to identify personalized effective immunotherapeutic strategies targeting T and B lymphocytes. Among the genetic variants associated with several autoimmune disorders, the C1858T polymorphism of the protein tyrosine phosphatase non-receptor type 22 (PTPN22) gene, encoding for Lyp variant R620W, affects the innate and adaptive immunity. We previously exploited a novel personalized immunotherapeutic approach based on siRNA delivered by liposomes (lipoplexes) that selectively inhibit variant allele expression. In this manuscript, we improved lipoplexes carrying siRNA for variant C1858T by functionalizing them with Fab of Rituximab antibody (RituxFab-Lipoplex) to specifically target B lymphocytes in autoimmune conditions, such as T1D. RituxFab-Lipoplexes specifically bind to B lymphocytes of the human Raji cell line and of human PBMC of healthy donors. RituxFab-Lipoplexes have impact on the function of B lymphocytes of T1D patients upon CpG stimulation showing a higher inhibitory effect on total cell proliferation and IgM+ plasma cell differentiation than the not functionalized ones. These results might open new pathways of applicability of RituxFab-Lipoplexes, such as personalized immunotherapy, to other autoimmune disorders, where B lymphocytes are the prevalent pathogenic immunocytes.

Simultaneous Inhibition of PD-1 and Stimulation of CD40 Signaling Pathways by Anti-PD-L1/CD40L Bispecific Fusion Protein Synergistically Activate Target and Effector Cells

Bispecific antibodies (BsAbs) or fusion proteins (BsAbFPs) present a promising strategy for cancer immunotherapy. Numerous BsAbs targeting coinhibitory and costimulatory pathways have been developed for retargeting T cells and antigen presenting cells (APCs). It is challenging to assess the potency of BsAb that engages two different signaling pathways simultaneously in a single assay format, especially when the two antigen targets are expressed on different cells. To explore the potency of anti-PD-L1/CD40L BsAbFP, a fusion protein that binds to human CD40 and PD-L1, we engineered CHO cells as surrogate APCs that express T cell receptor activator and PD-L1, Jurkat cells with PD-1 and NFAT-luciferase reporter as effector T cells, and Raji cell with NFkB-luciferase that endogenously expresses CD40 as accessory B cells. A novel reporter gene bioassay was developed using these cell lines that allows anti-PD-L1/CD40L BsAbFP to engages both PD-1/PD-L1 and CD40/CD40L signaling pathways in one assay. As both reporters use firefly luciferase, the effects of activating both signaling pathways is observed as an increase in luminescence, either as a higher upper asymptote, a lower EC50, or both. This dual target reporter gene bioassay system reflects potential mechanism of action and demonstrated the ability of anti-PD-L1/CD40L BsAbFP to synergistically induce biological response compared to the combination of anti-PD-L1 monovalent monoclonal antibody and agonist CD40L fusion protein, or either treatment alone. The results also showed a strong correlation between the drug dose and biological response within the tested potency range with good linearity, accuracy, precision, specificity and stability indicating properties, suggesting that this “three-cell-in-one” dual target reporter gene bioassay is suitable for assessing potency, structure-function and critical quality attributes of anti-PD-L1/CD40L BsAbFP. This approach could be used for developing dual target bioassays for other BsAbs and antibodies used for combination therapy.

A Metabolomics Investigation of the Metabolic Changes of Raji B Lymphoma Cells Undergoing Apoptosis Induced by Zinc Ions

Zinc plays a pivotal role in the function of cells and can induce apoptosis in various cancer cells, including Raji B lymphoma. However, the metabolic mechanism of Zn-induced apoptosis in Raji cells has not been explored. In this study, we performed global metabolic profiling using UPLC−Orbitrap−MS to assess the apoptosis of Raji cells induced by Zn ions released from ZnO nanorods. Multivariate analysis and database searches identified altered metabolites. Furthermore, the differences in the phosphorylation of 1380 proteins were also evaluated by Full Moon kinase array to discover the protein associated Zn−induced apoptosis. From the results, a prominent increase in glycerophosphocholine and fatty acids was observed after Zn ion treatment, but only arachidonic acid was shown to induce apoptosis. The kinase array revealed that the phosphorylation of p53, GTPase activation protein, CaMK2a, PPAR−γ, and PLA−2 was changed. From the pathway analysis, metabolic changes showed earlier onset than protein signaling, which were related to choline metabolism. LC−MS analysis was used to quantify the intracellular choline concentration, which decreased after Zn treatment, which may be related to the choline consumption required to produce choline-containing metabolites. Overall, we found that choline metabolism plays an important role in Zn-induced Raji cell apoptosis.

A Novel Peptide Derived from Ginger Induces Apoptosis through the Modulation of p53, BAX, and BCL2 Expression in Leukemic Cell Lines

Despite the efficacy of chemotherapy, the adverse effects of chemotherapeutic drugs are considered a limitation of leukemia treatment. Therefore, a chemotherapy drug with minimal side effects is currently needed. One interesting molecule for this purpose is a bioactive peptide isolated from plants since it has less toxicity to normal cells. In this study, we extracted protein from the Zingiber officinale rhizome and performed purification to acquire the peptide fraction with the highest cytotoxicity using ultrafiltration, reverse-phase chromatography, and off-gel fractionation to get the peptide fraction that contained the highest cytotoxicity. Finally, a novel antileukemic peptide, P2 (sequence: RALGWSCL), was identified from the highest cytotoxicity fraction. The P2 peptide reduced the cell viability of NB4, MOLT4, and Raji cell lines without an effect on the normal peripheral blood mononuclear cells. The combination of P2 and daunorubicin significantly decreased leukemic cell viability when compared to treatment with either P2 or daunorubicin alone. In addition, leukemic cells treated with P2 demonstrated increased apoptosis and upregulation of caspase 3, 8, and 9 gene expression. Moreover, we also examined the effects of P2 on p53, which is the key regulator of apoptosis. Our results showed that treatment of leukemic cells with P2 led to the upregulation of p53 and Bcl-2-associated X protein, and the downregulation of B-cell lymphoma 2, indicating that p53 is involved in apoptosis induction by P2. The results of this study are anticipated to be useful for the development of P2 as an alternative drug for the treatment of leukemia.

Antisense oligonucleotide p45Skp-2 suppresses migratory chemotactic and metastasis of oral malignant Burkitt’s lymphoma cell through down-regulation of MTA-1 and induction of E-cadherin mechanism

Introduction: Burkitt’s lymphoma is a high-grade B-cell neoplasm and one of the most aggressive malignancies of lymphoid origins which found mainly in the paediatric population. The treatment options of this tumour are still limited. However, a new strategy for refractory tumour, phosphorothioate oligonucleotide antisense technique has watched with keen interest. This study was aimed to examine the effect of antisense p45Skp-2 (Skp-2 AS) suppressed migratory chemotactic and metastasis of oral malignant Burkitt’s lymphoma (Raji) cell through down-regulation of MTA-1 and E-cadherin. Methods: True experiment laboratory with post-test control group design was confirmed in this study. The efficiency of Skp-2 AS in the suppression of cell chemotactic migration was examined by Boyden chamber assay. To evaluate the inhibition of cell metastasis was conducted by decreasing MTA-1 expression protein. The expressions of MTA-1, E-cadherin and α-tubulin protein were investigated by Western blot analysis. Results: The results revealed that the number of chemotactic migration of Skp-2 AS treated Raji cell was significantly decreased when compared with that of sense p45Skp-2 (Skp-2 S) and scrambled control (SC) cells (P<0.05) followed by decreased expressions of MTA-1 protein and overexpression of E-cadherin. Interestingly, the expression of α-tubulin protein as an internal control was approximately similar in each transfectant cells. Conclusion: p45Skp-2 have an antitumor activity via suppression of migratory chemotactic activity and metastasis on oral Burkitt’s lymphoma cells through down-regulation of MTA-1 and induction of E-cadherin proteins targeting this molecule could represent a promising new therapeutic approach for this type of cancer.

Downregulation of Cardiotrophin-Like Cytokine 1(CLCF1) Inhibits Osteoblastic Differentiation by Regulating RANKL/ OPG Pathway

BackgroundCardiotrophin-like cytokine factor 1 (CLCF1) is a member of the IL (interleukin)-6-type cytokine. Although its immunomodulatory functions are well defined, data on the physiological and pathological functions of the CLCF1 in bone metabolism remains scant. Here, we interrogated the functions and mechanisms of CLCF1 in osteoblast differentiation.MethodsA total of 109 patients with postmenopausal osteoporosis (PMOP) and 94 control group participants were included in this study. Quantitative reverse transcription PCR (RT-qPCR) and Western blot techniques were used to profile the expression of the CLCF1, nuclear factor-kB ligand and its decoy receptor osteoprotegerin (RANKL/OPG), as well as the janus activated kinase (JAK2)/transcription3 (STAT3) pathway. To elucidate the effect of CLCF1 on bone metabolism, we established CLCF1 gene-knockout Raji cell lines and Raji-MG-63 co-culture system, and interrogated the osteoblast differentiation by measuring the activity of ALP and the level of Alizarin-Red S staining.ResultsOur data demonstrated that the expression of CLCF1 was highly downregulated in PMOP compared with the control group. Moreover, the level of CLCF1 was proportional to the bone mineral density (BMD) in the postmenopausal osteoporosis. Furthermore, the deletion of CLCF1 gene inhibits osteoblast differentiation by regulating the RANKL/OPG system.ConclusionOur findings unravel the previously unidentified roles of CLCF1 in the bone-immune crosstalk, as well as potential therapeutic strategies for postmenopausal osteoporosis.

Anticancer potential of methanolic extracts from Pleurotus species on raji cells and antibacterial activity against Methicillin-Resistant Staphylococcus aureus

Prastiyanto ME, Rukmana RM, Saraswati DK, Darmawati S, Maharani ETW, Tursinawati Y. 2020. Anticancer potential of methanolic extracts from Pleurotus species on raji cells and antibacterial activity against Methicillin-Resistant Staphylococcus aureus. Biodiversitas 21: 5644-5649. The aim of this work is to identify the potential effects of methanolic extracts from four species of the Pleurotus genus cultivated in Indonesia on nasopharynx cancer (Raji cell line), and to investigate their antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA). This study investigates four species members of Pleurotus (Pleurotus ostreatus, P. cystidiosus, P. flabellatus, and P. pulmonarius var. stechangii). Dry Samples were extracted with methanol to yield crude extracts. Cytotoxicity screening was conducted using MTT assay of dry extracts, while antibacterial activity was calculated based on the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) using Mueller–Hinton broth, via the microdilution method. Compounds were analyzed using thin-layer chromatography (TLC). P. flabellatus provided the highest yield of dry extract (20.8%) with the lowest value of IC50 (556.226 µg/mL) compared to the three other species investigated. Antibacterial activity was calculated as MIC and MBC values against MRSA by the P. flabellatus extract which reached 6.25 mg/mL and 250 mg/mL, respectively. The result of TLC of the dry extract of P. flabellatus revealed the presence of terpenoids. P. flabellatus has the potential to be developed as both an anti-cancer and an antibacterial agent, especially against Raji cells and MRSA strains. However, further in vivo research and discovery of the modes of action involved are still needed to shed light on these effects. Studies can provide new information about the benefits of Pleurotus as a source of natural anticancer and antibacterial compound.

Preparation and Preliminary Evaluation of 68 Ga-Acridine: An Attempt to Study the Potential of Radiolabeled DNA Intercalator as a PET Radiotracer for Tumor Imaging

Introduction: Acridine is a well-known DNA intercalator and thereby gets easily inserted within DNA. As uncontrolled rapid cell division is one of the primary characteristics of the tumors, it is expected that acridine or its suitable derivatives will have preferential accumulation in the tumorous lesions. Therefore, an attempt was made to radiolabel an acridine derivative with 68Ga and study the potential of the 68Ga-acridine complex as a PET agent for tumor imaging. Methods: 9-aminoacridine was coupled with p-NCS-benzyl-DOTA to render it suitable for labeling with 68Ga. The purified acridine-DOTA conjugate was radiolabeled with 68Ga, eluted from a 68Ge/68Ga radionuclide generator. Various radiolabeling parameters were optimized and the stability of the radiolabeled preparation was studied. The biological behavior of the 68Ga-acridine complex was studied both in vitro and in vivo using Raji cell line and fibrosarcoma tumor bearing Swiss mice, respectively. Results: 68Ga-acridine complex was obtained with ~100% radiochemical purity under the optimized reaction conditions involving incubation of 2mg/mL of ligand at 100°C for 30 minutes. The complex maintained a radiochemical purity of >95% in normal saline and >65% in human blood serum at 3h post-incubation. In vitro cellular study showed (3.2±0.1)% uptake of the radiotracer in the Raji cells. Biodistribution study revealed significant tumor accumulation [(11.41±0.41)% injected activity in per gram] of the radiotracer within 1h postadministration along with uptake in other non-target organs such as, blood, liver, GIT kidney etc. Conclusion: The present study indicates the potential of 68Ga-acridine as a PET agent for imaging of tumorous lesions. However, further detailed evaluation of the agent is warranted to explore its actual potential.