Shangqin Liu

Inhibitory effect of baicalein on IL-6-mediated signaling cascades in human myeloma cells.

Interleukin‐6 (IL‐6) is an important growth factor for myeloma cells. IL‐6 promotes the survival and proliferation of multiple myeloma (MM) cells through the phosphorylated proteins, including STAT3, MAPK, and Akt. Chemical components that suppress the signaling proteins’ phosphorylation have a potential role for MM therapy. We recently identified that baicalein, a component of Scutellaria radix, suppressed proliferation and induced apoptosis of myeloma cells by blocking IκB‐α degradation followed by down‐regulating IL‐6 and XIAP gene expression. In the present study of four myeloma cell lines, namely U266, NOP2, AMO1, and ILKM2, we demonstrated that baicalein not only inhibited IL‐6‐mediated phosphorylation of signaling proteins, such as Jak, STAT3, MAPK, and Akt, but also inhibited the expression of their target genes, such as bcl‐xl. Finally, baicalein facilitated myeloma cell proliferation inhibited by dexamethasone. In contrast, baicalin, another major flavonoid derived from Scutellaria radix, had no significant effect on IL‐6‐mediated protein phosphorylation. Baicalein had no effect on Akt phosphorylation induced by the insulin‐like growth factor‐1 (IGF‐1) in NOP2 cells. Compared with PD98059, an MAPK inhibitor, baicalein exhibited a stronger inhibitory effect on Erk1/2 phosphorylation. Our results demonstrate that baicalein is a potent inhibitor of protein phosphorylation induced by IL‐6, and thus may be a useful agent for the treatment of MM.

Accelerated proliferation of myeloma cells by interleukin-6 cooperating with fibroblast growth factor receptor 3-mediated signals.

Interleukin-6 (IL-6) is a cytokine that regulates the proliferation of some tumor cells including multiple myeloma (MM). Ectopic expression of fibroblast growth factor receptor 3 (FGFR3) associated with the chromosomal translocation, t(4;14)(p16.3;q32), is frequently found in MM, and therefore, has been implicated in the neoplastic transformation of this disease. Here, we show that IL-6 together with FGF enhanced proliferation of a myeloma cell line, KMS-11 carrying t(4;14)(p16.3;q32) and the FGFR3-transfected U266 myeloma cell line which ectopically expressed FGFR3 but responded to neither IL-6 nor FGF alone. In KMS-11, IL-6 activated signal transducer and activator of transcription 3 (STAT3) while FGF activated extracellular signal-regulated kinase 1/2 (ERK1/2) and phosphatidylinositol (PI)-3 kinase. As both MEK inhibitors and a PI 3-kinase inhibitor abolished the effect of IL-6 and FGF, the activation of both the ERK1/2 and PI 3-kinase signaling cascades is essential for the proliferation of KMS-11 enhanced by IL-6 and FGF. Furthermore, the FGF-induced activation of ERK1/2 contributed to the serine phosphorylation of STAT3, suggesting that the signaling crosstalk between the cytokine receptor, IL-6 receptor α/gp130 and the growth factor receptor tyrosine kinase, FGFR3. These results indicate that FGFR3 plays a crucial role in the accelerated proliferation of MM carrying t(4;14)(p16.3;q32).

Interleukin-6, CD45 and the Src-Kinases in Myeloma Cell Proliferation

Abstract Multiple myeloma (MM) is a proliferative disorder of monoclonal plasma cells which accumulate in human bone marrow, and myeloma cells proliferate in response to a cytokine, interleukin-6 (IL-6). We recently found that MPC-1−CD49e− immature myeloma cells expressing CD45 form a proliferating population in MM. IL-6 activates at least two intracellular pathways including signal transducer and activator of transcription 3 (STAT3) and extracellular signal-regulated kinase 1/2 (ERK1/2) following the activation of Janus kinases (JAKs) via its receptor complexes composed of the IL-6 receptor α chain and gp130. Although the roles of CD45 have been extensively studied for antigen receptors in B and T cells, its physiological consequences in other hematopoietic cells remain largely unknown. Myeloma cells expressing CD45 antigens which contain the activation of src family protein-tyrosine kinases (PTKs) independent of IL-6 stimulation proliferate in response to IL-6, whereas the proliferation of CD45 cells which lack a considerable activity of the src family PTKs is not promoted by IL-6. The STAT3 and ERK1/2 pathways are similarly activated by IL-6 in both cells either expressing or not expressing CD45. In this review, we argue a novel mechanism of proliferation of myeloma cells, in that the activation of both STAT3 and ERK1/2 is not sufficient for IL-6-induced proliferation which further requires IL-6-independent activation of the src family kinases associated with CD45 phosphatase. We propose that the cellular context, such as CD45 expression and src family kinase activation, is crucial for myeloma cells to proliferate in response to IL-6.

GROWTH MECHANISM OF HUMAN MYELOMA CELLS BY INTERLEUKIN-6

Human myeloma cells are heterogenous morphologically and phenotypically. Myeloma cells can be classified into at least 5 subpopulations; MPC-1-CD45+CD49e-, MPC-1-CD45-CD49e- immature myeloma cells, MPC-1+CD45-CD49e-, MPC-1+CD45+CD49e- intermediate myeloma cells and MPC-1+CD45+CD49e+ mature myeloma cells. Interleukin-6(IL-6) is a major growth factor for human myeloma cells, but only MPC-1-CD45+CD49e- immature myeloma cells can response directly to IL-6 to proliferate. In the U-266 cell lines, IL-6 can lead to the induction of CD45 expression and CD45+ U-266 cells can proliferate in response to IL-6. In primary myeloma cells, MPC-1-CD45-CD49e-immature myeloma cells sorted from bone marrow samples can be changed to CD45+ cells by addition of IL-6 in vitro. In both CD45- and CD45+ U-266 cells, STAT3 and MAPK(ERK1/2) can be activated in response to IL-6 equally between them, but src family kinases such as Lyn, Fyn can be activated only in CD45+ U-266 cells. Thus, the activation of the src family kinases associated with CD45 expression is a prerequisite for the proliferation of myeloma cells. In the bone marrow of myeloma patients, most myeloma cells do not express CD45, and CD45+ immature myeloma cells are only 1∼2%. In order to clarify the difference of cellular context between CD45- and CD45+ myeloma cells, PCR-based cDNA subtraction was performed from CD45+ U-266 cells to CD45-U-266 cells. The series of this subtraction selected several genes. Furthermore, sensitivity to stress stimuli between CD45+ and CD45- U-266 cells was also compared. CD45-U-266 cells were markedly more resistant to stress conditions such as serum-free condition. Therefore, we can speculate that in the bone marrow of human myelomas IL-6 can induce proliferation of CD45+ immature cells, but the amount of IL-6 is too low to support CD45+ myeloma cells and loss of CD45 results in no direct response to IL-6 to proliferate but confers resistance to stress condition leading to the longer survival at the limited amount of IL-6.

Increased susceptibility to apoptosis in CD45(+) myeloma cells accompanied by the increased expression of VDAC1.

Expression of CD45 is quite variable in human myeloma cells and cell lines, such as U266, and CD45+ U266 proliferates in response to a growth factor, interleukin-6. Here, we show that CD45+ myeloma cell lines were more sensitive to various apoptotic stimuli, such as oxidative stress and endoplasmic reticulum (ER)-stress, than CD45− cells. Reactive oxygen species and calcium ion seemed to be involved in the susceptibility to apoptosis of CD45+ U266. The activation of the src family kinases associated with CD45 phosphatase played an important role in the augmented apoptosis in CD45+ U266 by oxidative stress. These results indicate that the CD45-expression renders myeloma cells competent for not only mitogenic but also apoptotic stimuli, resulting in either proliferation or apoptosis of CD45+ myeloma cells dependently upon the circumstantial stimuli. Furthermore, voltage-dependent anion channel (VDAC) 1 was identified as a gene highly expressed in CD45+ U266 by cDNA subtraction. The increased expression of VDAC1 seemed to augment the sensitivity to the ER-stress because the VDAC1-transfected U266 was more susceptible to the thapsigargin-induced apoptosis. Thus, CD45 expression accompanied by the increased VDAC1 expression sensitizes myeloma cells to the various extracellular stimuli that trigger apoptosis via the mitochondrial pathways.

Dehydroepiandrosterone can inhibit the proliferation of myeloma cells and the interleukin-6 production of bone marrow mononuclear cells from patients with myeloma.

The serum levels of an adrenal sex hormone, dehydroepiandrosterone sulfate (DHEA-S), are significantly more decreased in human myelomas compared with the reduction brought by physiologic decline with age. In order to clarify the effect of DHEA on myeloma cells, we investigated whether DHEA and DHEA-S could inhibit interleukin-6 (IL-6) production of bone marrow mononuclear cells and the proliferation of myeloma cells from patients with myeloma. DHEA-S and DHEA suppressed IL-6 production from a bone marrow stromal cell line, KM-102, as well as in bone marrow mononuclear cells from patients with myeloma. Furthermore, DHEA inhibited in vitro growth of the U-266 cell line and primary myeloma cells from the patients, as well as the in vivo growth of U-266 cells implanted i.p. in severe combined immunodeficiency-hIL6 transgenic mice. DHEA up-regulated the expression of peroxisome proliferator-activated receptor (PPAR), PPAR beta, but not PPARgamma or PPARalpha, and the expression of IkappaBalpha gene in myeloma cells and bone marrow stromal cells, which could explain the suppressive effect of DHEA on IL-6 production through the down-regulation of NF-kappaB activity. Therefore, these data revealed that DHEA-S, as well as DHEA, had a direct effect on myeloma and bone marrow stromal cells to inhibit their proliferation and IL-6 production, respectively.

Induction of Multilineage Markers in Human Myeloma Cells and Their Down-Regulation by Interleukin 6

Human primary myeloma cells are well known to be heterogeneous with regard to morphology and surface phenotype. We confirmed the heterogeneous expression of such multilineage markers as CD33, CD7, CD56, CD4, and CD86 in primary myeloma cells from 20 patients with multiple myeloma and in 8 human myeloma cell lines. CD33 expression in the Liu01 cell line, a subclone of U266 cells, and in vitamin D3-treated ILKM3 cells, correlated with a monocytoid morphology featuring convoluted nuclei and with increased C/EBPα expression. CD56+ myeloma cells from some myeloma patients and the CD56+ (but not the CD56-) myeloma cell lines expressed neuronal cell markers, such as neuron-specific enolase and β-tubulin III. CD7 expression in Liu01 cells and forskolin-stimulated U266 cells coincided with the presence of large cytoplasmic granules, and these cells featured increased expression of perforin messenger RNA and significant natural killer cell activity. Interleukin 6 (IL-6), a growth factor for myeloma cells, down-regulated CD33, CD7, or CD56 expression in primary myeloma cells, as well as in Liu01 cells. Therefore, these data suggest that human myeloma cells are capable of inducing the expression of multilineage markers and that IL-6 can down-regulate such expression.

CD19 Expression and Growth Inhibition of Tumours in Human Multiple Myeloma

Multiple myeloma (MM) is a proliferative disorder of monoclonal plasma cells which accumulate in human bone marrow (BM). CD19 is a hallmark differentiation antigen of the B cell lineage and positively regulates antigen receptor signal transduction in mature B cells. We have previously shown that malignant plasma cells (myeloma cells) isolated from the MM patients lack the CD19 expression, while non-malignant plasma cells isolated from the healthy donors do express the CD19 antigens. It is also intriguing that there exists both CD19 m and CD19 + plasma cells in some cases in pre-myeloma states including monoclonal gammopathy of undetermined significance (MGUS). It indicates that MGUS is usually composed of phenotypically non-malignant (CD19 + ) and malignant (CD19 m ) plasma cells. Furthermore, we recently demonstrate that, expression of the CD19 gene markedly inhibits the proliferation of human myeloma cell lines in vitro, and exhibits the reduced tumorigenicity in vivo and no anchorage-independent growth in vitro of a tumorigenic myeloma cell line. This inhibitory effect might result from the CD19-mediated intracellular signals because it is not observed in cells expressing the mutant CD19, which lacks the cytoplasmic domain. In this review, we suggest that loss of CD19 in MM could contribute to the proliferative advantage of the malignant plasma cell clones in this disease. Furthermore, we propose the usefulness of the phenotypic analysis of plasma cells in human plasma cell dyscrasia as a new diagnostic tool, and the CD19 gene as a potential target for the gene therapy in MM.

The regulatory mechanism of IL-6-dependent proliferation of human myeloma cells.

Abstract Multiple myeloma (MM) is a malignant tumor of plasma cells in the bone marrow. Interleukin 6 (IL-6) is an indispensable growth factor for myeloma cells. The heterogeneity of myeloma cells are the characteristics of MM, categorized into five sub-populations, two immature cells, MPC-1<PRE>-</PRE> CD49e<PRE>-</PRE> CD45<PRE>+/-</PRE>, intermediate cells, MPC-1<PRE>+</PRE> CD49e<PRE>-</PRE> CD45<PRE>+/-</PRE>, and mature cells, MPC-1<PRE>+</PRE> CD49e<PRE>+</PRE> CD45<PRE>+</PRE>. Only MPC-1<PRE>-</PRE> CD49e<PRE>-</PRE> CD45<PRE>+</PRE> immature cells (∼2% of total myeloma cells) respond to IL-6 to proliferate. CD45 protein tyrosine phosphatase is the determinant of IL-6 dependent cell growth of myeloma cells, although well studied IL-6 signal transducing factors, such as, IL-6Ra, gp130, Jak2, STAT3, and MAPK, are activated and involved in the process. Immature CD45<PRE>-</PRE> cells converted to CD45<PRE>+</PRE> cells after IL-6 stimulation both in U266 cells and sorted myeloma cells from the bone marrow aspirates of MM patients. CD45<PRE>-</PRE> cells are relatively resistant to serum starvation compared to CD45<PRE>+</PRE> cells. Because IL-6 level in the bone marrow is low even in MM patients, the CD45<PRE>-</PRE> phenotype of myeloma cells may protect the cells from apoptosis. These findings of a tuning effect of CD45 on myeloma cell proliferation may aid the study of IL-6 dependent proliferation of myeloma cells and lead to the development of new therapies for MM patients.