Gm40600 suppressed SP 2/0 isograft tumor by reducing Blimp1 and Xbp1 proteins.

BACKGROUND: Multiple myeloma (MM), characterized by cancerous proliferation of plasmablasts (PB) and plasma cells (PC), remains incurable in many patients. Differentially expressed molecules between MM PCs and healthy PCs have been explored in order to identify novel targets for treating MM. In the present study, we searched for novel MM therapeutic targets by comparing mRNA expression patterns between the Mus musculus myeloma plasmablast-like SP 2/0 cell line and LPS-induced PB/PC. METHODS: Gene expression profiles of LPS-induced PB/PC and SP 2/0 cells were determined using RNA-sequencing. A predicted gene (Gm40600) was found to be expressed at a low level in SP 2/0 cells. To study the role of Gm40600 in malignant PC, Gm40600 cDNA was cloned into a lentiviral vector (LV201) containing a puromycin selectable marker that was then transfected into SP 2/0 cells. Stable Gm40600-expressing SP 2/0 cells were selected using puromycin. The effect of Gm40600 on SP 2/0 cell proliferation, cell cycle/apoptosis, and tumor progression was assessed by cell counting kit-8 (CCK8), flow cytometry (FACS), and the SP 2/0 isograft mouse model, respectively. The effect of Gm40600 on mRNA and protein expression was evaluated by RNA-sequencing and western blotting, respectively. RESULTS: We found that SP 2/0 cells expressed lower level of Gm40600 mRNA as compared to LPS-induced PB/PC. Overexpression of Gm40600 significantly suppressed SP 2/0 cell proliferation and isograft tumor progression in an isograft mouse model by promoting apoptosis. In addition, Gm40600 overexpression suppressed transcription of the gene encoding Bcl2. Gm40600 overexpression also reduced the expression of PC-associated transcription factors Blimp1 and Xbp1, which promote transcription of the gene that encodes Bcl2. CONCLUSIONS: Gm40600 reduced SP 2/0 cell proliferation and isograft tumor growth and progression by suppressing Blimp1 and Xbp1-mediated Bcl2 transcription to induce apoptosis. Thus, regulation of a human homolog of Gm40600, or associated factors, may be a potential therapeutic approach for treating MM.

Hypericin-mediated photodynamic therapy induces apoptosis of myoloma SP2/0 cells depended on caspase activity in vitro.

BACKGROUND: Photodynamic therapy (PDT) is becoming a promising therapeutic modality for hematological malignancies. Hypericin is a natural photosensitizer possessing anti-depressant, anti-virus and anti-cancer activities. The present study was designed to explore the effect and mechanism of hypericin-mediated PDT on the mouse multiple myeloma (MM) cells in vitro. METHODS: The mouse myeloma SP2/0 cells were incubed with different concentrations of hypericin and then illuminated with different light doses. The inhibitory effect of hypericin-mediated PDT on tumor cell proliferation was assayed by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) method. The apoptosis related morphological changes of SP2/0 cells were observed by microscopy. The biochemical hallmarks of apoptosis such as DNA fragments, mitochondrial membrane potential changes were assessed. The expression of apoptosis related proteins were investigated by western blotting. RESULTS: Hypericin-mediated PDT induced the proliferation inhibition and apoptosis of tumor cells in a dose dependent manner. Tumor cells showed obvious morphological changes of apoptosis and necrosis and DNA fragmentation after treated by hypericin mediated PDT (0.025 ~ 0.05 µM). The mitochondria membrane potential in SP2/0 cells was decreased significantly after incubated with the 0.025 µM and 0.5 µM hypericin (P < 0.05). The expression level of caspase-3 was decreased, while caspase activity was elevated with the increasing drug dosage. The apoptosis of SP2/0 cells was blocked by a pan-caspase inhibitor Z-VAD-FMK and caspase-3 inhibitor Ac-DEVD-CHO. CONCLUSION: Hypericin-mediated PDT induced apoptosis mainly dependent on caspase related pathways. Hypericin-mediated PDT may be a potential and alternative therapy for MM.

Enhancing Antibody-Specific Productivity: Unraveling the Impact of XBP1s Overexpression and Glutamine Availability in SP2/0 Cells.

Augmentation of glycoprotein synthesis requirements induces endoplasmic reticulum (ER) stress, activating the unfolded protein response (UPR) and triggering unconventional XBP1 splicing. As a result, XBP1s orchestrates the expression of essential genes to reduce stress and restore homeostasis. When this mechanism fails, chronic stress may lead to apoptosis, which is thought to be associated with exceeding a threshold in XBP1s levels. Glycoprotein assembly is also affected by glutamine (Gln) availability, limiting nucleotide sugars (NS), and preventing compliance with the increased demands. In contrast, increased Gln intake synthesizes ammonia as a by-product, potentially reaching toxic levels. IgA2m(1)-producer mouse myeloma cells (SP2/0) were used as the cellular mammalian model. We explored how IgA2m(1)-specific productivity (qIgA2m(1)) is affected by (i) overexpression of human XBP1s (h-XBP1s) levels and (ii) Gln availability, evaluating the kinetic behavior in batch cultures. The study revealed a two and a five-fold increase in qIgA2m(1) when lower and higher levels of XBP1s were expressed, respectively. High h-XBP1s overexpression mitigated not only ammonia but also lactate accumulation. Moreover, XBP1s overexpressor showed resilience to hydrodynamic stress in serum-free environments. These findings suggest a potential application of h-XBP1s overexpression as a feasible and cost-effective strategy for bioprocess scalability.

Loc108167440 suppressed myeloma cell growth by P53-mediated apoptosis.

Multiple myeloma (MM) results from biased proliferation of cancerous plasma cells (PC). Therapeutic strategies that target MM PC will provide immense value to the treatment of MM. For this, it is necessary to identify novel molecules that differ between MM PC and healthy PC. RNA sequencing was used to determine differences in gene expression profiles between LPS-induced plasmablasts (PB)/PC and the PB-like myeloma SP 2/0 cell line. Compared to LPS-induced PB/PC, SP 2/0 cells expressed significantly lower levels of Loc108167440 mRNA. Loc108167440 overexpression reduced the number of SP 2/0 cells by stimulating apoptotic cell death. In addition, Loc108167440 overexpression suppressed tumor progression in the SP 2/0 xenograft mouse model. Finally, we demonstrated that Loc108167440 overexpression up-regulated expression of p53 in SP 2/0 cells. These results suggest that Loc108167440 overexpression suppressed SP 2/0 cell growth by inducing p53-mediated apoptosis. Thus, Loc108167440 overexpression may be a potential therapy for treating MM.

Effect of Cysteamine on Cell Growth and IgG4 Production in Recombinant Sp2.0 Cells.

The manipulation of redox potential in secretory pathway by thiol reducing agents can be a strategy to improve the production levels of disulfide-bonded proteins including recombinant antibodies. Here we have studied the influence of cysteamine on viability and the production level of IgG4 in Sp2.0 cells. For this purpose, the recombinant Sp2.0 cells producing an anti CD33 IgG4, were subjected to different concentrations of cysteamine. At concentrations of 2, 4 and 5 mM cysteamine, the secreted levels of IgG4 did not change significantly. However, in concentration of 7 mM cysteamine, a significant decrease was observed in IgG4 levels which may indicate the cytotoxicity of this compound in higher concentrations. Our results show that the cysteamine treatment reduces the cell viability in a dose-dependent manner. Also it was observed that 2 mM cysteamine had no late effect on IgG4 production level and only at day 3, this concentration of cysteamine decreased the cell viability significantly. To test whether the addition of cysteamine can affect the expression level of protein disulfide isomerase, RT-PCR analysis was carried out. The results revealed that cysteamine does not affect the PDI transcription and expression level of IgG4 in this type of recombinant cells.