[Changes of Serum Complement C1q in Patients with Multiple Myeloma].

OBJECTIVE: To analyze the change of serum C1q in the course of multiple myeloma (MM) and its correlation with clinical characteristics. METHODS: A total of 138 newly diagnosed MM patients in Zhongnan Hospital of Wuhan University from June 2016 to December 2019 were selected as research objects, during the same period 50 age-matched anemia patients, 50 lymphoma patients, 50 leukemia patients, and 50 myelodysplastic syndrome (MDS) patients were selected as control groups. All the patients met WHO disease classification, and were definitely diagnosed by pathology or bone marrow smear/biopsy. The changes of C1q between MM patients and control group, as well as in different therapeutic responses of MM patients before and after treatment were compared, also the difference of clinical characteristics among MM patients with different C1q level, so as to analyze risk factors which led to C1q decline. RESULTS: The average value of C1q in MM patients was (128.18±51.24) mg/L, which was significantly lower than control group (P<0.01). The levels of white blood cell, platelet (PLT), hemoglobin (Hb), serum calcium, albumin, lactate dehydrogenase (LDH) in newly diagnosed high C1q group were significantly higher than those in low C1q group (P<0.05). Logistic analysis showed that the levels of PLT, Hb, albumin, and LDH in newly diagnosed high C1q group were higher than those in low C1q group (r=0.248, r=0.394, r=0.405, r=0.295). After treatment, the levels of C1q in MM patients with complete remission and very good partial remission were significantly higher than before treatment (P<0.05), while those with partial remission and stable disease also increased but not significantly (P>0.05). CONCLUSION: The C1q level in MM patients is significantly lower than that in patients with other hematologic system diseases, and it increases with the remission of the disease after treatment.

Baicalein Inhibits Proliferation of Myeloma U266 Cells by Downregulating IKZF1 and IKZF3.

BACKGROUND Baicalein can suppress the growth of multiple tumors, including multiple myeloma (MM), but the exact mechanisms remains elusive. Here, we investigated the exact mechanisms of the anti-myeloma activity of baicalein. MATERIAL AND METHODS Proliferation and rates of apoptosis of myeloma U266 cells exposed to baicalein were detected. Microarray, polymerase chain reaction (PCR) assay, and Western blot analysis were applied to evaluate the mRNA and protein levels of associated molecules. Survival analysis of IKZF1 and IKZF3 was conducted as well. RESULTS Baicalein suppressed the growth and stimulated apoptosis of myeloma U266 cells in a dose- and time-dependent way. Baicalein increased mRNA level of CRBN, and further studies suggested that baicalein downregulated IKZF1 and IKZF3 on a post-transcriptional level. Although the differences did not reach statistical significance, IKZF1 and IKZF3 were associated with poor overall survival. CONCLUSIONS Our results suggest that baicalein suppresses the growth and promotes apoptosis of myeloma U266 cells through downregulating IKZF1 and IKZF3. Baicalein increased the expression of CRBN, which might exert a reversion effect on resistance of IMiDs. MM patients in IKZF1 and IKZF3 low-expression groups had better overall survival than those in IKZF1 and IKZF3 high-expression groups. Thus, the present results indicate that baicalein might be a therapeutic choice for targeting IKZF1 and IKZF3.

Expression of cancer-testis antigen in multiple myeloma.

Recently, the immunotherapy has been highlighted among cancer treatments. Cancer-testis antigen (CTA) has been studied in a variety of solid tumors because of its specific expression in tumors, and testis, ovary and placenta tissues, but not in other normal tissues. In order to provide a new approach for multiple myeloma (MM) immunotherapy, we examined the CTA expression in MM cell lines, and primary myeloma cells in patients with MM. Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to detect the mRNA expression of MAGE-C1/CT7, SSX1, SSX2 and SSX4 in MM cell lines of RPMI-8226 and U266, and bone marrow (BM) cells of 25 MM patients and 18 healthy volunteers. The results showed that the 4 CTAs were expressed in RPMI-8226 and U266 cell lines. The positive expression rate of MAGE-C1/CT7, SSX1, SSX2 and SSX4 in the BM cells of 25 MM patients was 28% (7/25), 80% (20/25), 40% (10/25) and 68% (17/25), respectively. In contrast, the expression of any member of the CTAs was not detected in BM cells of 18 healthy volunteers. The expression of two or more CTAs was detected in 80% (20/25) MM patients, and that of at least one CTA in 88% (22/25). The mRNA expression levels of SSX1 and SSX4 were significantly higher in patients with MM at stage III than in those at stage I and II (P<0.05). No statistically significant differences were observed in the mRNA expression levels of MAGE-C1/CT7 and SSX2 in further stratified analyses by age, gender, MM types and percentage of MM cells in BM (P>0.05). In conclusion, our present study showed that MAGE-C1/CT7, SSX1, SSX2 and SSX4 were co-expressed in MM cell lines and the primary myeloma cells in MM patients, but not expressed in BM cells of healthy subjects. The mRNA levels of SSX1 and SSX4 are associated with MM clinical stage. This work may provide a new insight into MM immunotherapy in the future.

[Effect of baicalein on proliferation and migration in multiple myeloma cell lines RPMI 8226 and U266 cells].

OBJECTIVE: To investigate the effect of baicalein on proliferation and migration of multiple myeloma (MM) cell lines and its molecular mechanism. METHODS: The MM cell line RPMI-8226 and U266 cells were used as the model, and treated with different concentration and time of baicalein the effect of baicalein on the MM cells proliferation was assessed by MTT assay. With or without baicalein or Interleukin-6 (IL-6) treatment, the ß-catenin protein level was analyzed by immunofluorescence assay and western blot assay and mRNA levels of ß-catenin, c-myc, cyclin D1 and integrin 7 gene by RT-PCR. Transwell chamber migration assay was used to detect the cells migration ability with different concentration of baicalein cultured. RESULTS: Baicalein inhibited the MM cell line RPMI 8226 and U266 cell proliferation in a dose- and time-dependent manner. It simultaneously inhibited ß-catenin protein level to resist the effect of IL-6 on inducing MM cell proliferation, and resulted in decrease of ß-catenin, c-myc, cyclinD1 and integrin ß7 mRNA levels. Baicalein also decreased migration ability of MM cells in a dose-dependent manner by SDF-1. CONCLUSION: Baicalein can inhibit MM cells proliferation and migration, and its molecular mechanisms are associated with inhibition of proliferation related genes ß-catenin, c-myc, cyclin D1 and integrin ß7 expression.

[Relationship between CD45 molecule expression and apoptotic sensitivity of myeloma cells].

OBJECTIVE: To investigate the effects of CD45 expression on induction of apoptosis in multiple myeloma cells. METHODS: Melphalan was used to induce myeloma cell line U266 apoptosis. Serum-free culture was used to induce CD45RB gene or empty plasmid transfected U266 apoptosis. The glucose-free culture was used to induce high CD45 (CD45(hi)) or low CD45 (CD45(low)) expression AMO1 apoptosis. Intraperitoneal inoculation was used to compare the survival of CD45(-) or CD45(+) U266 cells in mice. The number of apoptotic cells and mitochondrial membrane potential (MMP) was detected by flow cytometry. Western blotting was used to detect the cytochrome C release from mitochondrial and caspase-9 activation. RESULTS: Melphalan treatment induced 45% of CD45(+) and 30% of CD45(-) U266 cells apoptosis. Compared with the CD45(low) AMO1 cells, CD45(hi) cells were more susceptible to apoptosis. In serum-free culture for five days, 60% of CD45RB transferred U266 cells underwent apoptosis, while in the empty plasmid transfected ones, apoptotic cell number was not significantly increased. The survival time of CD45(-) U266 cells in the SCID-hIL-6 mice was 5 times that of CD45(+) cells. After melphalan treatment, 60% of the CD45(+) U266 cells lost MMP, while only 30% of CD45(-) U266 cells, and 10% of control cells did so. After UV irradiation, CD45(+) U266 cells mitochondria released more cytochrome C, leading to more caspase-9 activation. CONCLUSION: CD45 expression is involved in mitochondria-mediated apoptotic process and increases apoptotic sensitivity of myeloma cells under a variety of stimulation.