KM3 Cells Research Articles

IL-6 Regulates the Chemosensitivity of Drug-Resistant Multiple Myeloma Cell Lines to Bortezomib through STAT3/Notch Signaling Pathway

To investigate the effect of interleukin-6 (IL-6) on the chemosensitivity of drug-resistant multiple myeloma (MM) cell lines to bortezomib (BTZ) and its mechanism. Peripheral blood samples were collected from patients with BTZ-resistant MM before and after treatment. Human MM cell lines KM3 and KM3/BTZ were cultured in vitro. ELISA was used to detect the content of IL-6 in peripheral blood of MM patients, KM3 and KM3/BTZ cells. CCK-8 assay was used to detect the drug sensitivity of KM3 and KM3 / BTZ cells to BTZ. KM3 / BTZ cells were divided into KM3/BTZ control group (normal culture for 48 h), IL-6 neutralizing antibody Anti-IL-6 group (500 ng/ml Anti-IL-6 treated for 48 h), BTZ group (300 ng/ml BTZ treated for 48 h), BTZ + Anti-IL-6 group (300 ng/ml BTZ and 500 ng/ml Anti-IL-6 treated for 48 h). The proliferation activity of KM3 / BTZ cells was detected by CCK-8 assay. The cell cycle distribution of KM3/BTZ cells was detected by flow cytometry. The apoptosis of KM3/BTZ cells was detected by Annexin V-FITC/PI double staining. The mRNA expression levels of IL-6, Notch1, signal transducer and activator of transcription 3 (STAT3) in KM3/BTZ cells were detected by real-time fluorescent quantitative PCR (qRT-PCR), and the protein expression levels of IL-6, Notch1, STAT3 in KM3/BTZ cells were detected by Western blot. The level of IL-6 in peripheral blood of patients with BTZ-resistant MM after treatment was significantly higher than that before treatment (P<0.05). The level of IL-6 in KM3/BTZ cells was significantly higher than that in KM3 cells (P<0.05). The sensitivity of KM3/BTZ cells to BTZ was significantly lower than that of KM3 cells (P<0.05), and the resistance index (RI) was 19.62. Anti-IL-6 and BTZ could inhibit the proliferation of KM3 / BTZ cells, block cell cycle, and induce apoptosis (P<0.05). Compared with single drug treatment, the combined effect of Anti-IL-6 and BTZ was more obvious on KM3/BTZ cells (P<0.05), and significantly down regulated the mRNA and protein expression of IL-6, Notch1 and STAT3 in KM3/BTZ cells (P<0.05). Antagonizing IL-6 can increase the chemosensitivity of MM cells to BTZ, and IL-6 may reduce the sensitivity of MM cells to BTZ through STAT3/Notch signaling pathway.

Design, synthesis and biological evaluation of tyrosine derivatives as Mcl-1 inhibitors

The upregulation of the protein myeloid cell leukemia-1 (Mcl-1) is closely associated with various human cancers, which can result in the evasion of apoptosis and a low survival rate. Therefore, developing Mcl-1 inhibitors has become a promising paradigm for cancer therapy. Herein, we designed and synthesized a novel series of tyrosine derivatives, among which compounds 5g, 6l and 6c exhibited very high binding affinity to Mcl-1 with Ki values of 0.18, 0.27 and 0.23 μM, respectively. Interestingly, compound 6l showed not only potent activity against Mcl-1 but also considerable selectivity over Bcl-2 and Bcl-xL, which was rationalized by molecular docking and fragment-centric topographical mapping (FCTM). It is worth noting that compounds 5g, 6l and 6c displayed potent antiproliferative activity against several cancer cell lines and could induce apoptosis of KM3 and HepG2 cells in a dose-dependent manner.

PB2104 INHIBITION OF NUCLEOLIN REVERSED BORTEZOMIB RESISTANCE VIA THE REGULATION OF NF‐κB PATHWAY IN MULTIPLE MYELOMA CELLS

Background:The proteasome inhibitor bortezomib has remarkable efficacy against multiple myeloma (MM). However, intrinsic and acquired resistance to bortezomib could limit its efficacy. The potential resistance mechanisms have yet to be clarified. The nucleo‐cytoplasmic protein nucleolin (NCL) is high expression in several tumors, especially in resistance cells, that may act in the drug resistance. Previously we first observed the higher expression of NCL in KM3 bortezomib resistant cells (KBTZ) than its parental KM3 MM cells, indicating the role of NCL in bortezomib resistance and needing further research to elucidate.Aims:To investigate the potential mechanism(s) by which NCL regulates bortezomib resistance in MM cell line.MethodsKM3 MM cells and its bortezomib resistant cells KBTZ were transfected with lentiviruses carrying the shRNA sequences that target the endogenous NCL gene and control scrambled gene respectively. Post‐transfected cells were named as followed: KM3‐NCL‐Knockdown(KM3‐NCL‐KD), KM3‐NCL‐Scramble(KM3‐NCL‐Scr), KBTZ‐NCL‐Knockdown(KBTZ‐NCL‐KD), KBTZ‐NCL‐Scramble(KBTZ‐NCL‐Scr). Effect of NCL on cell viability and cells drug sensitivity were measured. Cell cycle related proteins and drug resistance related molecules expression level were evaluated. Related signaling pathways were also examined by western blot.Results:NCL silencing significantly inhibited cell proliferation and colony formation ability in KM3 and KBTZ cells. Colony sizes and numbers were both decreased in NCL silenced cells. Colony formation rates in KM3‐NCL‐KD, KM3‐NCL‐Scr, KBTZ‐NCL‐KD, KBTZ‐NCL‐Scr were 9.33% ± 0.47%, 27.22% ± 1.85%, 7.16% ± 0.24% and 28.22% ± 3.57%, respectively (P &lt; 0.05). Moreover, NCL silencing arrested cell cycle transition in G0/G1, resulted in an increasing trend in cell apoptosis. CyclinD, cyclinE, CDK2 proteins were downregulated and P21 protein was upregulated after knockdown of NCL. Notably, NCL silencing increased the sensitivity to bortezomib in KM3 cells and prominently reversed the resistance to bortezomib in KBTZ cells. IC50of bortezomib were 17.29 ± 3.35 ng/mlin KM3‐NCL‐Scr cells and 10.13 ± 1.5 ng/mlin KM3‐NCL‐KD cells (P &lt; 0.05), indicating a 1.7‐fold increase of bortezomib sensitivity after NCL knockdown. IC50of bortezomib were 590.78 ± 147.92 ng/mlin KBTZ‐NCL Scr cells and 63.48 ± 13.16 ng/mlin KBTZ‐NCL KD cells (P &lt; 0.05), meaning a 10.8‐fold reversal of bortezomib resistance after NCL knockdown (Figure 1). Besides, compared with Scr cells, both KM3‐ and KBTZ‐NCL‐KD cells displayed lessrhodamine‐123 effluxion, suggesting that NCL silencing suppressed drug efflux activity. Knockdown of NCL declined ATP‐binding cassette transporter proteins MRP1, LRP, BCRP at mRNA levels and protein levels. Defined mRNA expression level of KM3‐NCL‐Scr and KBTZ‐NCL‐Scr cells equal to 1, relative mRNA expression of MRP1, LRP, BCRPwere 0.56 ± 0.26, 0.13 ± 0.03, 0.13 ± 0.09 in KM3‐NCL‐KD cells and 0.69 ± 0.11, 0.35 ± 0.07, 0.53 ± 0.20 in KBTZ‐NCL‐KD cells, respectively. Furthermore,downregulated expression of P‐NF‐κB, P‐IκBα, IKKα and P‐IKKα protein, decreased intranuclear NF‐κB level and upregulated expression of IκBα protein were observed in both KM3‐ and KBTZ–NCL‐KD cells, indicating that NCL may act by regulating activity of NF‐κB signaling pathway.Summary/Conclusion:In conclusion, our resultssuggested that inhibition of NCL reversed bortezomib resistance via the regulation of NF‐κB pathway in multiple myeloma cells, indicatingthe novel molecular understanding of bortezomib resistance and the role of NCL in MM resistance, and the emerging marker of NCL as a potential target.image

Effect of Bushen Yanggu Decoction on Multidrug Resistance of Multiple Myeloma Cell Line KM3/BTZ

To explore the effect of Bushen Yanggu Decoction (BYD) on drug resistance and proliferation of human multiple myeloma-resistant KM3/BTZ cells. Human multidrug-resistant KM3/BTZ cells were established by Bortezomib (BTZ) gradient induction. The effects of commonly used chemotherapeutic drugs and serum containing Bushen Yanggu Decoction (BYD) on the proliferation of KM3 cells and KM3/BTZ cells were detected by MTT assay. RT-qPCR and Western blot were used to detect the expression of Par-4, HSP27 and P-gp genes. Flow cytometry was used to detect cell apoptosis. The established KM3/BTZ cells could produce varying degree of resistance to commonly used chemotherapeutic drugs. Among them, the highest resistance index (RI) to BTZ was 20.269. MTT assay showed that the proliferation of KM3/BTZ cells treated with serum containing Bushen Yanggu Decoction was inhibited, and the inhibitory effect increased with the serum concentration incranse of Bushen Yanggu Decoction. The serum containing Bushen Yanggu Decoction could inhibit the proliferation of KM3/BTZ cells, and induce apoptosis, significantly reduce the drug-resistance of KM3/BTZ cells, up-regulate the expression of Par-4, down-regulate the expression of HSP27 and P-gp. Bushen Yanggu Decoction can effectively inhibit the proliferation of KM3/BTZ cells and induce apoptosis. Bushen Yanggu Decoction can effectively reverse the multidrug-resistance of KM3/BTZ cells. The mechanism may be related with the decrease of expression of HSP27 and P-gp and the increase of expression of Par-4.

Triptolide Induces Cell Apoptosis by Targeting H3K4me3 and Downstream Effector Proteins in KM3 Multiple Myeloma Cells.

As the principal active ingredient in the Chinese herb Tripterygium wilfordii Hook.F (TwHF), triptolide has been shown to have very strong antitumor properties. The trimethylation of lysine 4 on histone H3 (H3K4me3) has been proposed to promote gene expression, and the accumulation of H3K4me3 at the transcriptional start sites of oncogenes is involved in carcinogenesis. To identify the association between the reduction of H3K4me3 and the apoptosis of MM cells induced by triptolide, we investigated the global patterns of H3K4me3 occupancy in the MM cell genome. Combined analyses using ChIP-on-chip and western blotting showed that H3K4me3 were highly enriched on the gene promoters of c-Myc and VEGFA and were associated with the up-regulation of both genes. Treatment of KM3 cells with triptolide and siRNA targeting ASH2L reduced the expression of c-Myc and VEGFA. These results suggest that triptolide can down-regulate c-Myc and VEGFA expression by blocking the accumulation of H3K4me3 on their promoters,and thus play an important role in anti-MM mechanism.

The Establishment of Bortezomib Resistant Myeloma Cell Line KM3/BTZ and Explore the Resistance Mechanism

Background: Multiple myeloma (MM) is an incurable B-cell malignancy resulting in significant morbidity and mortality, the incidence of second place in hematological malignancies. The proteasome inhibitor bortezomib inhibits IkappaB degradation, prevents NF-kappaB activation, and induces apoptosis in MM cells, has become first-line drug to MM. Despite its promising activity in traditional chemoresistant myeloma patients, however, some patients are resistant or become refractory to bortezomib. Chauhan D et al reported that Blockade of Hsp27 overcomes bortezomib resistance in lymphoma cells (Chauhan D et al., Cancer Re, 2003). However, bortezomib resistance mechanisms in MM remain controversial, the molecular basis of this reduced responsiveness is currently not fully understood.Objective: To establish a bortezomib-resistant cell line KM3 /BTZ of MM, then search for genes related to bortezomib resistance phenotype by analyzing the differential gene expression patterns with cDNA microarray,and explain the mechanism of bortezomib-resistant multiple myeloma.Methods: Bortezomib-resistant KM3/BTZ cell line was generated by increasing concentration of bortezomib to induce KM3 cells in vitro. MTT assay was employed to detect the cytotoxic effect on KM3 and KM3/BTZ cells. The cells apoptosis were analyzed by flow cytometry. We detect the gene expression profile changes between KM3/BTZ and its parent cell line KM3 cells by CDNA microarray. Combining of molecular annotation system MAS3.0 software and detailed analysis of documented resistance genes were used to analyze the data. Specific differentially-expressed genes were chosen for further verification using real-time RT-PCR. The expression of multi-drug resistance 1(MDR-1) gene mRNA was determined by RT-PCR.Results: The KM3/BTZ cell line was established successfully. Bortezomib resistance index values were significantly higher in KM3/BTZ (IC50 =351.2±3.51ng /ml) when compared to KM3 cells (IC50 = 17.8±1.03ng /ml),and the resistance index is 19.7 (P< 0.05). Through gene expression profiling filtering, compared with KM3,770 significantly differently expressed genes were screened out in the KM3/BTZ cell line, of which up and down regulated genes were 287 and 383 respectively, in which heat shock protein family gene HSPB2 was significantly up-regulated (Ratio=14.7455). Downregulated gene list in KM3/BTZ cell line included transcriptional regulators like ZNF family proteins (Tab 1). Upregulated genes in KM3/BTZ cell line included signal transduction related genes like MS4A family protein (Tab 1) also transcriptional regulators like ZNF family proteins (Tab 1). The expression trend of eight genes for further verification was almost consistent with the microarray, excepting gene of JUN (Fig 1). The expression of MDR-1 mRNA was not observed in either resistant or parental cells (Fig 2).Conclusion: We have successfully established bortezomib-resistant cell line KM3/BTZ. MDR-1 was not involved in bortezomib-resistant multiple myeloma cells.HSPB2, ZNF and MS4A family genes are probably related to bortezomib resistance in KM3/BTZ cells. Our results suggest that they may be new targets to overcome resistance to bortezomib in patients with MM.Table 1ZNF and MS4A family gene expression in KM3/BTZ and KM3 cellsGeneB signal*B detection*A signal*A detection*BvsA signal log ratio*BvsA change*ZNF71185.08P206.16P0.41269DZNF926.75P14.57P0.46328DZNF70410.7A26.78P0.39955DZNF4927.08P3.48P2.03448IZNF53287.93P43.54A2.01952IZNF59410.25P4.63P2.21382IZNF789269.79P97.3P2.77276IZNF506330.36P149.48P2.21006IMS4A31721.58P145.3P11.84845IMS4A4A2688.23P100.8P26.66895IMS4A6A141.96P56.47M2.513901IMS4A149.02P53.11P0.92299NC PHigh expression; A: Low expression; M:Marginally D: Decrease; I: Increase; NC: No change*: A:KM3; B: KM3/BTZ [Display omitted] [Display omitted] MDNA Marker; 1-2: K562/A cell line; 3-4: KM3cell line; 5-6: KM3/BTZ cell line DisclosuresNo relevant conflicts of interest to declare.

Cryopreserved mouse fetal liver stromal cells treated with mitomycin C are able to support the growth of human embryonic stem cells.

An immortalized mouse fetal liver stromal cell line, named KM3, has demonstrated the potential to support the growth and maintenance of human embryonic stem cells (hESCs). In this study, the characteristics of KM3 cells were examined following cryopreservation at −70°C and in liquid nitrogen for 15, 30 and 60 days following treatment with 10 μg/ml mitomycin C. In addition, whether the KM3 cells were suitable for use as feeder cells to support the growth of hESCs was evaluated. The inhibition of mitosis without cell death was observed when the KM3 cells were treated with 10 μg/ml mitomycin C for 2 h. The morphology of the KM3 cells cryopreserved in liquid nitrogen for 60 days was not markedly changed, and the cell survival rate was 84.60±1.14%. By contrast, the survival rate of the KM3 cells was 66.40±2.88% following cryopreservation at −70°C for 60 days; the cells readily detached, were maintained for a shorter time, and had a reduced expression level of basic fibroblast growth factor. hESCs cultured on KM3 cells cryopreserved in liquid nitrogen for 60 days showed the typical bird’s nest structure, with clear boundaries and a differentiation rate of 16.33±2.08%. The differentiation rate of hESCs cultured on KM3 cells cryopreserved at −70°C for 60 days was 37.67±3.51%. These results indicate that the cryopreserved KM3 cells treated with mitomycin C may be directly used in the subculture of hESCs, and the effect is relatively good with −70°C short-term or liquid nitrogen cryopreservation.

Serum deprivation elevates the levels of microvesicles with different size distributions and selectively enriched proteins in human myeloma cells in vitro

To investigate the effects of serum deprivation (SD) on microvesicles (MVs) secreted from human myeloma cells and the implications for disease progression. RPMI 8226, U266, and KM3 human myeloma cells were incubated in medium containing 10% (non-SD) or 1% fetal bovine serum (SD) and MVs were isolated. The levels and size distribution of MVs were analyzed with flow cytometry. The protein profiles of MVs were studied using 2D SDS-PAGE, MALDI-TOF-MS, and Western blotting. NF-κB activation was analyzed using EMSA. Angiogenesis was examined in Eahy926 endothelial cells. Exposure of RPMI 8226 cells to SD for 24 h did not alter the number of apoptotic cells. However, SD increased the number of MVs from RPMI 8226, U266, and KM3 cells to 2.5-, 4.3-, and 3.8-fold, respectively. The size distribution of SD MVs was also significantly different from that of non-SD MVs. Three proteins ZNF224, SARM, and COBL in SD MVs were found to be up-regulated, which were involved in cell cycle regulation, signal transduction and metabolism, respectively. Co-culture of SD MVs and RPMI 8226 cells increased NF-κB activation in the target RPMI 8226 cells. Furthermore, SD MVs from RPMI 8226 cells significantly increased the microtubule formation capacity of Eahy926 endothelial cells compared with non-SD MVs. SD elevates the levels of microvesicles with different size distribution and selectively enriched proteins in human myeloma cells in vitro. The selectively enriched proteins, especially ZNF224, may play key roles in regulation of myeloma cells, allowing better adaptation to SD.

Human umbilical cord blood-derived stromal cells: A new resource for the proliferation and apoptosis of myeloma cells

Background/ObjectiveBone marrow stromal cells (BMSCs) can support multiple myeloma (MM) disease progression and resistance to chemotherapy. The proliferation of MM cells may be suppressed by modifying the hematopoietic microenvironment (HME). We have previously isolated human umbilical cord blood-derived stromal cells (hUCBDSCs) and observed that hUCBDSCs suppressed proliferation and induced apoptosis in KM3 cells. To examine the mechanism by which hUCBDSCs drive the inhibition of MM, KM3 cells were co-cultured with hUCBDSCs.MethodsInterleukin (IL)-6 and soluble IL-6 receptor (sIL-6R) expression levels were measured by enzyme-linked immunosorbent assay. The expression levels of membrane IL-6 receptor (mIL-6R), intercellular cell adhesion molecule-1 (ICAM-1), B-cell lymphoma/leukemia-2 (Bcl-2), and Bcl-XL as well as the location of nuclear factor κB (NF-κB) were assessed by laser confocal microscopy. The expression profiles of mIL-6R and ICAM-1 were also more precisely examined by flow cytometry, and Bcl-2, Bcl-XL and inhibitor kappa B expression levels were analyzed by western blot. The mRNA expression levels of IL-6R, ICAM-1, Bcl-2, and Bcl-XL were assessed by real-time polymerase chain reaction. NF-κB DNA-binding activity was examined by electrophoretic mobility shift assay.ResultsThe protein expression levels of both sIL-6R and mIL-6R were reduced in culture conditions when KM3 cells were co-cultured with hUCBDSCs; moreover, the mRNA expression levels of IL-6R were also reduced. Nuclear translocation of the NF-κB p65 subunit was inhibited in KM3 cells by co-culture with hUCBDSCs. Moreover, hUCBDSCs inhibited NF-κB DNA-binding activity, thereby resulting in the downregulation of NF-κB-regulated proteins.ConclusionhUCBDSCs can suppress proliferation and induce apoptosis in KM3 cells by both downregulating IL-6R expression and inhibiting NF-κB activity.

Effects of down-regulated TRAF6 gene expression on the proliferation and apoptosis in multiple myeloma cells

To investigate the down-regulated TRAF6 gene expression and its effects on proliferation and apoptosis in multiple myeloma (MM) cells. Detection of TRAF6 expression were conducted by RT-PCR and Western blot in MM cell lines of KM3, U266, RPMI8226 and primary cells from patients. RPMI8226 cell lines were transfected with siRNA of TRAF6. The efficiency of transfection was identified by using of fluorescence microscope, RT-PCR, and Western blot. The levels of proliferation were analyzed by CCK-8 method under the different concentrations of siRNA. Apoptosis rate were detected with Hoechst33258/PI double staining by flow cytometry. Apoptosis related proteins Bcl-2, BAX, and NF-κB signal pathway were observed before and after siRNA transfection by Western blot. The levels of TRAF6 mRNA and protein in MM cell lines, especially in primary myeloma cells, were significantly higher than those in controls. After transfected with 50 nmol/L siRNA in RPMI8226 cells, the relative level of TRAF6 mRNA (0.49±0.24) was significantly lower than that in non-transfected group (1.87±0.23) and idling group (1.74±0.35). The proliferation rate of siRNA transfected cells decreased with dose dependence (P<0.01). The apoptosis rates increased from 11.20% (before transfection) to 51.82% (after transfection), accompanied by down-regulated Bcl-2 protein, NF-κB signal pathway (p-p65 and p52), and up-regulated BAX protein. TRAF6 expression was high in myeloma cells. TRAF6 siRNA could inhibit proliferation of myeloma cells and induce apoptosis mediated by NF-κB classical and alternative pathway in myeloma cells.

Epigenetic inactivation of ADAMTS9 via promoter methylation in multiple myeloma

A disintegrin‑like and metalloprotease with thrombospondin typeⅠ motifs(ADAMTS) are a family of 19secreted mammalian metalloproteases. ADAMTS9 was reported to be a novel tumor suppressor gene and is downregulated in various types of human cancer due to hypermethylation of promoter CpG islands. In the present study, the silencing mechanism of the ADAMTS9 gene was analyzed in the multiple myeloma (MM) cell lines, KM3 and RPMI‑8226. Control and MM samples were obtained by conventional bone marrow (BM) biopsy of normal and MM adult BM, respectively. RT‑PCR revealed a high expression of the ADAMTS9 gene in normal samples and RPMI‑8226 cells while marked gene silencing of ADAMTS9 was observed in MM patients and KM3 cells. Promoter methylation of ADAMTS9 was detected in the KM3 cell line and 66% (37/56) MM patients by methylation‑specific PCR. In addition, the DNA demethylating agent, 5‑aza‑2'‑deoxycytidine and trichostatinA restored ADAMTS9 expression by suppressing promoter methylation in KM3 cells. Ectopic expression of ADAMTS9 in ADAMTS9‑silenced MM cells was found to significantly suppress cell colony formation and proliferation. In the present study, DNA methylation was found to play a key role in ADAMTS9 gene silencing and the biological behavior of myeloma cells. The results demonstrate that ADAMTS9 silencing by methylation may be a novel tumor marker for MM and the applicability of demethylating agents in the treatment of MM.

Lycorine induces cell-cycle arrest in the G0/G1 phase in K562 cells via HDAC inhibition

BackgroundLycorine, a natural alkaloid extracted from Amaryllidaceae, has shown various pharmacological effects. Recent studies have focused on the potential antitumor activity of lycorine. In our previous study, we found that lycorine decrease the cell viability of leukemia HL-60 cells and multiple myeloma KM3 cells and induces cell apoptosis. However, the effect and molecular mechanism of lycorine on human chronic myelocytic leukemia cells has yet to be determined.MethodsHuman chronic myelocytic leukemia cells K562 were treated with lycorine. Cell viability was monitored using the method of CCK-8. The histone deacetylase (HDAC) enzymatic activity was detected by HDAC colorimetric assay, and the cell cycle was analyzed by flow cytometry. The expression of cell-cycle related proteins were identified using Western blot.ResultsIn the present study, we further revealed that lycorine can inhibit the proliferation of K562 cells. Analysis of HDAC activity showed that lycroine decreases HDAC enzymatic activities in K562 cells in a dose-dependent manner. Inhibition of HDAC activity has been associated with cell-cycle arrest and growth inhibition. We evaluated the cell cycle distribution after lycorine treatment and found that lycorine causes cell-cycle arrest in the G0/G1 phase. To investigate the mechanism behind this cell cycle arrest, G1-related proteins were assayed by Western blot. After lycorine treatment, cyclin D1 and cyclin-dependent kinase 4 expressions were inhibited and retinoblastoma protein phosphorylation was reduced. Lycorine treatment also significantly upregulated the expression of p53 and its target gene product, p21.ConclusionsThese results suggest that inhibition of HDAC activity is responsible for at least part of the induction of cell-cycle arrest in the G0/G1 phase by lycorine and provide a mechanistic framework for further exploring the use of lycorine as a novel antitumor agent.

Betulinic acid inhibits autophagic flux and induces apoptosis in human multiple myeloma cells in vitro

To investigate the effects of betulinic acid (BA) on apoptosis and autophagic flux in multiple myeloma cells and the relationship between the two processes. The proliferation of human multiple myeloma KM3 cells was measured with MTT assay. FITC/PI double-labeled flow cytometry (FCM) and Hoechst 33258 staining were used to analyze the cell apoptosis. Caspase 3, PARP, Beclin1, LC3-II, and P62 were detected using Western blotting. Treatment of KM3 cells with BA (5-25 μg/mL) suppressed the cell proliferation in time- and dose-dependent manners. The IC(50) values at 12, 24, and 36 h were 22.29, 17.36, and 13.06 μg/mL, respectively. BA treatment dose-dependently induced apoptosis of KM3 cells, which was associated with the activation of caspase 3. However, Z-DEVD-FMK, a specific inhibitor of caspase 3, did not decrease, but rather sensitized the cells to BA-induced apoptosis, suggesting an alternative mechanism involved. On other hand, BA treatment dose-dependently increased the accumulation of LC3-II and P62 in KM3 cells, representing the inhibition of autophagic flux. Furthermore, BA treatment dose-dependently downregulated the expression of Beclin 1, an important inducer of autophagy, in KM3 cells. In the presence of BA, Z-DEVD-FMK induced autophagy and increased the amount of LC3-II in KM3 cells, which may occur via attenuating BA-induced decrease in the level of Beclin 1. Similarly, rapamycin, an autophagy inducer, increased the amount of LC3-II in KM3 cells. In the presence of BA, rapamycin caused further increase in the amount of LC3-II. Furthermore, rapamycin sensitized BA-treated KM3 cells to apoptosis. The results demonstrate that BA induces apoptosis and blocks autophagic flux in KM3 cells. Furthermore, in addition to activation of caspase 3, the inhibition of autophagic flux also contributes to the BA-mediated apoptosis of KM3 cells.

Immortalized mouse fetal liver stromal cells support growth and maintenance of human embryonic stem cells

Human embryonic stem cells (hESCs) are usually maintained in an undifferentiated state by co-culture with feeder cells. The feeder cells are important for the growth of hESCs. A novel spontaneously immortalizated mouse fetal liver stromal cell line, named KM3, was isolated from a13.5‑day mouse fetal liver. In this study, we examined whether KM3 cells could be used as feeders to support the growth of hESCs. hESCs cultured on KM3 cells showed a similar proliferation rate and characteristics to mouse embryonic fibroblasts(MEFs) after prolonged culture, including morphology, unlimited and undifferentiated proliferative ability, maintenance of normal karyotypes, formation of embryoid bodies invitro and typically immature teratomas invivo. Our results indicate that the immortalized KM3 cell line has the potential to support the growth and maintenance of hESCs. The cell line may be used for the large-scale expansion of hESCs in a low-cost and less labor-intensive manner.

BAFF‐R gene induced by IFN‐γ in multiple myeloma cells is related to NF‐κB signals

B-cell activating factor (BAFF) is a potent cell-survival factor expressed in many haematopoietic cells. BAFF regulates B-cell survival, differentiation and proliferation by binding to three tumour necrosis factor receptors: transmembrane activator, calcium modulator and cyclophilin ligand interactor; B-cell maturation antigen; and BAFF receptor (BAFF-R). Although BAFF-R is produced by interferon gamma (IFN-γ), the underlying mechanism remains elusive. In this study, we examined the effects of IFN-γ on BAFF-R expression in cultured human multiple myeloma cells (KM3) both at the transcriptional and posttranscriptional levels. Incubation of KM3 cells with IFN-γ elevated the expression of BAFF-R mRNA and protein levels. IFN-γ elicited marked enhancement of the human BAFF-R promoter activity and nuclear factor kappa B (NF-κB) DNA binding activity. NF-κB dependent on the human BAFF-R gene might be regulated via a transcriptional event through one putative NF-κB site on the BAFF-R gene promoter. These results provide a molecular mechanism for the increase in expression of the BAFF-R gene that is induced by proinflammatory cytokines in responsive cells.

Discovery of chrysoeriol, a PI3K-AKT-mTOR pathway inhibitor with potent antitumor activity against human multiple myeloma cells in vitro

This study was designed to determine the impact of chrysoeriol on proliferation and cell cycle progression in the human multiple myeloma cell lines RPMI 8226 and KM3, and its related molecular mechanisms. Chryseoriol was identified by using the phosphorylated AKT-specific cytoblot high throughput assay. CCK-8 assay was employed to examine the growth inhibition rate and IC(50) (48 h) in peripheral blood mononuclear cells (PBMNCs), RPMI 8226 and KM3 cells treated with chrysoeriol at various concentrations. Cells were labeled with 5-6-carboxyfluorescein diacetate succinimidyl ester (CFSE), and the proliferation dynamics was detected by flow cytometry and analyzed with ModFit software. The cell cycles of RPMI 8226 and KM3 cells were measured by flow cytometry when the IC(50) concentration of chrysoeriol was adopted. The alterations in cell-cycle related proteins (Cyclin B1, Cyclin D1, p21) and proteins in PI3K-AKT-mTOR pathway were determined by Western blot analysis. The results showed the proliferation of multiple myeloma cells was significantly inhibited by chrysoeriol, resulting in cell cycle arrest in G(2)/M phase. Chrysoeriol could significantly reduce the expression of p-AKT (s473) and p-4eBP1 (t37/46) protein, meanwhile enhanced Cyclin B1 and p21 protein expression. Similar effects were not observed in PBMNCs from normal donors. It was concluded that chrysoeriol was a selective PI3K-AKT-mTOR pathway inhibitor. It restrained the proliferation of human multiple myeloma cells, but didn't affect proliferation of PBMNCs from normal donors. It might exhibit the cell cycle regulatory effect via the inhibition of PI3K-AKT-mTOR signal pathway.

Effect of human recombinant PDCD5 protein on cell apoptosis of multiple myeloma KM3 cells induced by dexamethasone and its mechanism

To observe the effect of programmed cell death 5 (PDCD5) protein on the apoptosis of multiple myeloma KM3 cells induced by dexamethasone and to understand its mechanism. The human recombinant PDCD5 (rhPDCD5) protein was added (alone of different concentrations or associated with dexamethasone) into KM3 cells. Cultured together for certain time, the cells were collected for the following experiments: (1)The effect of rhPDCD5 protein and dexamethasone on the apoptotic rate of KM3 cells was determined by flowcytometry (FCM) analysis after the cells were stained by Annexin V-FITC & PI (propidium iodide). (2)Caspase-3 activity of KM3 cells was evaluated by Western blot. (3)The expression of survivin protein in KM3 cells was detected by immunocytochemistry. The apoptotic rate of KM3 cells and the activity of caspase-3 increased significantly, and that treated with rhPDCD5 protein and dexamethasone was higher than that treated with rhPDCD5 protein only. The expression of survivin protein in the rhPDCD5 with dexemethas group was down-regulated, and with the concentration of rhPDCD5 and dexamethasone increasing, the changes was more obviously. PDCD5 protein can induce the apoptosis of KM3 cells, and accelerate the apoptosis of KM3 cells induced by dexamethasone. PDCD5 protein may reduce the expression of survivin protein and increase activation of caspase-3 to play its role in promoting apoptosis.

Berbamine, a novel nuclear factor κB inhibitor, inhibits growth and induces apoptosis in human myeloma cells

We sought to investigate the effect of berbamine on the growth of human multiple myeloma cell line KM3 and elucidate the mechanism of its action. MTT assay was used to determine the inhibitory effect of berbamine alone or combined with chemotherapeutic drugs. Flow cytometry was performed to characterize cell cycle profile in response to berbamine treatment. Western blot was used to measure the protein levels of p65, IkappaB Kinase alpha (IKKalpha), TNFAIP3 (A20), IkappaBalpha, p-IkappaBalpha, cyclinD1, Bcl-2, BAX, Bcl-x(L), Bid, and survivin. Berbamine inhibits the proliferation of KM3 cells in a dose- and time-dependent manner. Combination of berbamine with dexamethasone (Dex), doxorubicin (Dox) or arsenic trioxide (ATO) resulted in enhanced inhibition of cell growth. Flow cytometric analysis revealed that KM3 cells were arrested at G(1) phase and apoptotic cells increased from 0.54% to 51.83% for 36 h. Morphological changes of cells undergoing apoptosis were observed under light microscope. Berbamine treatment led to increased expression of A20, down-regulation of IKKalpha, p-IkappaBalpha, and followed by inhibition of p65 nuclear localization. As a result, NF-kappaB downstream targets such as cyclinD1, Bcl-x(L), Bid and survivin were down-regulated. Berbamine inhibits the growth of KM3 cells by inducing G(1) arrest as well as apoptosis. Berbamine blocks NF-kappaB signaling pathway through up-regulating A20, down-regulating IKKalpha, p-IkappaBalpha, and then inhibiting p65 nuclear translocation, and resulting in decreased expression of the downstream targets of NF-kappaB. Our results suggest that berbamine is a novel inhibitor of NF-kappaB activity with remarkable anti-myeloma efficacy.

Regulation of B-lymphocyte stimulator expression levels in response to IFN-γ and IL-6

Objective To investigate the regulation of B-lymphocyte stimulator(BLyS) levels in response to IFN-γand IL-6. Methods Flow cytometry, quantitative polymerase chain reaction, ELISA and Western blot were applied to examine the expression level of BLyS in response to IFN-γ and IL-6 . Results IFN-γand IL-6 induced BLyS expression in KM3 cells. After treated with BAY11-7082, an IkB-α phospho- rylation inhibitor, the up regulation of BI,yS induced by IFN-γ was completely inhibited. Inhibiting the nu-clear faetor-kB (NF-kB) and mitogen activated protein kinase(MAPK) activation in KM3 cells reduced BLyS protein and gene expression. Conclusion MAPK and NF-kB pathways are involved in the regulation of BLyS expression, which suggests that MAPK and NF-kB might be used for the treatment of multiple mye- loma. Key words: Multiple myeloma; B-lymphocyte stimulator; Nuclear faetor-kB; Mitogen activatedprotein kinase

B‐Lymphocyte stimulator: a new biomarker for multiple myeloma

Multiple myeloma (MM) is a common malignant tumor, characterized by unlimited proliferation of abnormal plasmocytes in bone marrow. Considering the biological function of B-Lymphocyte stimulator (BLyS) and its receptors in B cell, we examined BLyS and its receptors expression in MM cells. Our studies confirmed that BLyS and its receptors are expressed in MM cells, including KM3, CZ-1, and primary MM cells, playing an important role in the survival and proliferation of MM cells. Additionally, we provide evidence that BLyS protein is located in the MM cell plasma membrane. We also found that IFN-gamma and IL-6 can induce BLyS expression on MM cells, while after the treatment of BAY11-7082, an IkB-alpha phosphorylation inhibitor, IFN-gamma induced up regulation of BLyS was completely inhibited, suggesting that nuclear factor kappaB (NF-kappaB) might be involved in the mechanism of the regulation of BLyS levels in response to cytokines. Finally, linear correlation analysis of the Lactate Dehydrogenase concentration and beta 2-microglobulin level with BLyS, and expressions of BLyS mRNA in MM patients revealed a significant correlation between them (P < 0.01 in all case), showing that BLyS could be a biomarker for the diagnosis and treatment of MM.