Disulfiram May Contribute to Apoptosis of Myeloma Cells By Reactivating of Cell Cycle Regulating Gene CDKN2A

Objective: Multiple myeloma (MM) is an incurable plasma-cell dyscrasia characterized by uncontrolled growth of malignant plasma cells in the bone marrow. Proteasome inhibitor bortezomib is a frontline MM drug, however acquired resistance to bortezomib remains a clinical hurdle. Disulfiram (tetraethylthiuram disulfide, DSF) belongs to promising cancer-killing drugs, and although the mechanism of its anticancer activity remains unclear, it has been suggested that this drug inhibits proteasome activity. Furthermore, disulfiram contains strong thio-reactive functional groups and since the catalytic mechanism of DNA methyltransferases (DNMTs) involves the covalent attack at the C6 position of cytosine by the thiol group of the catalytic cysteine on the DNMT enzyme, the DSF can function as DNMT non-nucleoside inhibitor with possible gene demethylation´s effect. On the other hand, DNMT inhibitors, 5-azacytidine (AZA) and 5-aza-2´-deoxycytidine (DAC) are nucleoside analogs, whose mechanism of action involves incorporation of the aza-modified base into DNA during DNA synthesis with subsequent covalent trapping of the DNMT enzyme. Therefore, these nucleoside analogs (AZA and DAC) can have significant cytotoxicity and can lead to major adverse effects, including myelosuppression, when administered to patients. Methods: RPMI8226 and U266 myeloma cells were exposed to DSF (0, 1 µM and 1 µM), DAC (0, 5 µM, 3, 75 µM and 5 µM), JNJ-7706621 (0, 5 µM and 5 µM) and 5 µM SAHA (suberoylanilide hydroxamic acid). Subsequently, we assessed for: i/ cell viability assay using 3-(4, 5dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide agent (MTT); ii/ DNA content by flow cytometry analysis; iii/ apoptosis determination by Annexin V and activity of caspase-3 and -7 quantification by cleavage of fluorogenic substrate CellEvent™ Caspase-3/7 Green Detection Reagent; iii/ CDKN2A and CDKN2B methylation status by methylation-specific PCR and pyrosequencing; iiii/ CDKN2A and CDKN2B gene expression analysis by quantitative RT-PCR. Results: To determine the influence of DSF, DAC and two cyclin dependent kinase (CDK) inhibitors JNJ-7706621 and SAHA on cell cycle regulation, RPMI8226 and U266 cell lines were treated with the same scheme as for methylation and expression analysis. U266 cell treatments with 1 µM DSF (18, 9 %) and 5 µM JNJ (21, 6 %) show similar parameters in sub-G1 cell population, but lower when they compared to SAHA treatment (29, 7 %). Analysis of apoptosis of cells treated with 1 µM DSF show 18,51 % of apoptotic cells, while 26,22 % and 28,4 % apoptotic cells after 5 µM SAHA and 5 µM JNJ treatments were detected. The proportions of cells in apoptosis in both 0, 1 µM and 1 µM DSF treatments are 21,8 % and 18,5 %. We have found unmethylated 162 bp CDKN2B analysed gene region, while 150 bp analysed CDKN2A gene region is methylated. Unmethylated state of the CDKN2B gene was confirmed by pyrosequencing of 223 bp promoter and 138 bp 1.exon gene regions. Expression of the unmethylated CDKN2B gene is not influenced, while the 0,1 µM DSF treatment causes increased expression of the CDKN2A gene (Graph1). Conclusion: In treated myeloma cells, we have examined apoptotic effect of DSF in comparison to CDK inhibitors JNJ-06621 and SAHA through detection of methylation and expression status of CDKN2A and CDKN2B genes. Our results indicate DSF pro-apoptotic effect comparable to used CDK inhibitors. The 5-aza-2´-deoxycytidine with demethylation activity and DSF do not affect an expression of the unmethylated CDKN2B gene, its expression effectively increases CDK inhibitors (Graph2). On the other hand, the expression of the methylated CDKN2A gene is only slightly increased in DSF treated cells in comparison to other treatments. This study was supported in part by NV18-03-0050 from the Ministry of Health of the Czech Republic and LF_2018_001 from Palacky University Olomouc. Figure. Figure. Disclosures No relevant conflicts of interest to declare.

Epinephrine Stimulates Cell Proliferation and Induces Chemoresistance in Myeloma Cells through the β-Adrenoreceptor in vitro

Objectives: To explore the effect of the β-adrenoreceptor signaling pathway on myeloma cells. Methods: The myeloma U266 cell line was treated with epinephrine and propranolol. Cell proliferation was analyzed by MTS assay. Apoptosis was detected by flow cytometry. The β-receptor subtype and the key enzyme of epinephrine were identified by reverse transcription polymerase chain reaction (RT-PCR). Results: Epinephrine (5-50 μM) promoted U266 cell growth in a dose-dependent manner and neutralized the inhibition effect of bortezomib (25 and 50 ng/mL) in vitro. Cell proliferation was inhibited by a β-receptor antagonist, propranolol, at a concentration of 50-200 μM. The proportions of early and late apoptotic cells were enhanced after treatment with propranolol. The expression of caspase 3/7, 8, and 9 was elevated in propranolol-treated myeloma cells. Both β1- and β2-adrenoceptor mRNAs were expressed in the U266 cell line. Key enzymes dopamine hydroxylase and tyrosinehydroxylase were identified in myeloma cells. Conclusions: Our results reveal that epinephrine stimulates myeloma cell growth in vitro while the β-blocker propranolol has an antiproliferative effect, indicating that stress hormones may trigger the progression of myeloma.

Anti-Cancer Effects of Clofazimine As a Single Agent and in Combination with Cisplatin in Multiple Myeloma

Multiple myeloma (MM) is a malignant neoplasm of bone marrow plasma B cells with high morbidity. Clofazimine (CLF) is an FDA-approved leprostatic, anti-tuberculosis, and anti-inflammatory drug that was previously shown to have growth suppression effects on various cancer types such as hepatocellular, lung, cervix, esophageal, colon, and breast cancers as well as melanoma, neuroblastoma, and leukemia cells. The objective of this study was to evaluate the anticancer effect of CLF on U266 resistant MM cell line. The relative cell viability of a panel of hematological cell lines (Jurkat, U266, Namalwa, K562, HL60) treated with 10 µM CLF after 24 h of treatment significantly reduced the viability in all cell lines, with percentages ranging between 28% (U266) and 38% (Jurkat) (p<0.001). IC50 value of CLF was found as 9.8 ± 0.7 µM on the U266 cell line. Previous studies showed that this level of CLF does not inhibit growth of healthy cells, which supports safety of CLF. CLF had both dose (2, 5, 10 µM) and time (12, 24, and 48 h) dependent growth inhibitory effect. Combination chemotherapy is an approach to increase the effectiveness of chemotherapeutics as well as overcome drug resistance and suppresses side effects of drugs. Therefore, we evaluate the combination effect of CLF in U266 cells and showed that combination with cisplatin led to a synergistic interaction between two compounds in all tested dose regimes, resulting in a 2.5-7.1 fold marked increase in cell death. Importantly this synergism was observed in U266 cells, which have mutant p53 at A161T showing resistance to cytotoxic agents such as platinum analogs (cisplatin etc.). <>Depolarization of the mitochondrial membrane is one of the first events in apoptosis. JC-1 is a lipophilic and cationic dye that reversibly changes color from green to red as the mitochondrial membrane potential increases (depolarization). JC-1 assay used in both flow cytometry analyses and fluorescence microscopy images have shown that relative to the control, CLF treatment results in the depolarization of mitochondrial membrane 15, 20.5, 14.3 fold respectively at 12, 24, and 48 h in U266 cell line (Figure 1). The caspase family of cysteine proteases plays an important role in apoptosis. Caspase-3 is a major protease activated during the early stages of programmed cell death. 10 µM CLF was applied for 12, 24, and 48 h and anti-active caspase-3 PE stained U266 cells were analyzed by flow cytometry. Caspase-3 activity is enhanced 5.6, 24.5 and 13.6-fold relative to untreated controls at 12h, 24h and 48 h respectively. Phosphatidylserine (PS) translocation to the outer leaflet of the cellular membrane is one of the key steps in early stages of apoptosis. To support our previous findings on apoptotic effect of CLF, we employed Annexin-V assay. CLF treatment caused a significant increase in the percentage of early and late apoptotic cells at 12 h (2.1 and 1.8 fold respectively), 24 h (4.1 and 12.3 fold) and 48 h (10.1 and 11.5 fold). Fluorescence microscopy images also supported flow cytometry data (Figure 2). Collectively, all three apoptosis assay results show that CLF significantly induces apoptosis in U266 cells. Our study is the first to show apoptotic and growth inhibitory effects of CLF on a p53-mutant resistant MM cell line U266. Our results also proved that combined therapy employing CLF together with chemotherapeutics seems to be a possible future therapeutic approach for MM. Further in vivo and clinical studies are warranted to evaluate its therapeutic potential for resistant MM treatment. Figure 1 Effect of 10 µM CLF on mitochondrial membrane potential. Flow cytometry fluorescence intensity A) Dot plots B) Bar plots of cells stained with JC-1 (n=3). C) Fluorescence microscopy image of JC-1-stained untreated cells indicating healthy mitochondria (red), D) In CLF-treated cells, green color shows diffusion of JC-1 from damaged mitochondria. Figure 1. Effect of 10 µM CLF on mitochondrial membrane potential. Flow cytometry fluorescence intensity A) Dot plots B) Bar plots of cells stained with JC-1 (n=3). C) Fluorescence microscopy image of JC-1-stained untreated cells indicating healthy mitochondria (red), D) In CLF-treated cells, green color shows diffusion of JC-1 from damaged mitochondria. Figure 2 Flow cytometry analysis of Annexin V-PE/7-AAD stained U266 cells treated with 10 µM CLF. A) Representative dot plots of Annexin V-PE vs 7-AAD signals gated as live, early apoptotic and late apoptotic quadrants B) Cell population bar graphs of corresponding dot plot quadrants (n=3). C) Early apoptotic U266 cell (right) stained with Annexin V-PE (green) and a late apoptotic U266 cell (left) stained with both Annexin V-PE (green) and nuclear dye PI (red) D) Close-up micrograph (160X) of a late apoptotic U266 cell. Figure 2. Flow cytometry analysis of Annexin V-PE/7-AAD stained U266 cells treated with 10 µM CLF. A) Representative dot plots of Annexin V-PE vs 7-AAD signals gated as live, early apoptotic and late apoptotic quadrants B) Cell population bar graphs of corresponding dot plot quadrants (n=3). C) Early apoptotic U266 cell (right) stained with Annexin V-PE (green) and a late apoptotic U266 cell (left) stained with both Annexin V-PE (green) and nuclear dye PI (red) D) Close-up micrograph (160X) of a late apoptotic U266 cell. Disclosures No relevant conflicts of interest to declare.

In Vitro JAK1/2 Inhibition, in Association with Bortezomib and Lenalidomide, Is Comparable with Bortezomib, Lenalidomide and Dexametasone: An Alternative for Multiple Myeloma Patients with JAK2 overexpression?

Background: Despite the absence of JAK1 and JAK2 mutations in multiple myeloma (MM), high levels of IL-6 constitutively activate the JAK/STAT pathway promoting survival and proliferation of tumor cells. Therefore, pharmacological inhibition of JAK proteins can be a potentially therapeutic strategy for myeloma treatment. Aims: 1) to identify expression of JAK1 and JAK2 in MM cell lines and in recently diagnosed MM patients; 2) to perform functional in vitro studies in MM cell lines treated with JAK/STAT pathway inhibitor (ruxolitinib), associated with drugs currently used in MM first line treatment (bortezomib, lenalidomide and dexamethasone), with and without co-culture with normal stromal cells; 3) to evaluate global gene expression of JAK/STAT pathway in cell lines treated with ruxolitinib to elucidate its mechanism of action in MM. Methods: JAK1 and JAK2 expression were analyzed in four cell lines (RPMI-8226, U266, SKO-007 and SKM-M2) and in bone marrow samples from 30 MM patients and 3 healthy controls by real time PCR. After IC50 calculation, drugs concentrations were: bortezomib (B) 10 nM for both RPMI-8226 and U266 cell lines; ruxolitinib (R) 30 µM for RPMI-8226 and 40 µM for U266 cell lines; lenalidomide (L) 10 µM for both cell lines; and dexamethasone (D) 1 µM for both cell lines. Apoptosis and cell cycle were evaluated by flow cytometry. PCR array for 92 JAK/STAT pathway related genes (Taqman® Array Human JAK/STAT Pathway, Applied Biosystems, Foster City, CA, USA) was performed in RPMI-8226 and U266 wild type and B+R treated cell lines, in duplicates. Results: Among the four cell lines, U266 presented the highest expression of JAK1 and JAK2 genes. JAK1 was overexpressed in 27% and JAK2 in 57% of 30 MM patients (considering at least 2-fold increase). After B+R treatment, RPMI-8226 showed increased number of cells in SubG0 phase (p<0.001) with reduction of cells in S (p<0.01) and G2/M (p<0.001) phases. In U266 cell line, there is a slight increase of cells in SubG0 phase (p<0.05). Also, after B+R treatment, both RPMI-8226 and U266 presented 50% of cells in late apoptosis, which was accompanied by reduction of expression levels of BCL-2 and BCL-XL anti-apoptotic genes. The expression profile of JAK/STAT pathway after B+R treatment showed that many JAK/STAT, Ras/Raf/MAPK and PI3K/Akt/mTOR pathways genes lost their expression, mainly in RPMI-8226, with insignificant changes in U266 expression pattern. Co-culture of RPMI-8226 with normal stromal cell line HS5 protected tumor cells from apoptosis, as the number of cells in late apoptosis decreased from 50% to 32% (p<0.001). The addition of immunomodulatory drug lenalidomide to the schedule (B+R+L) increased tumor cell death from 32% to 73% in co-culture (p<0.001). Despite the impressive results, B+R+L schedule was equivalent to currently used treatment for standard risk MM patients B+L+D (67% of cell death, p>0.05), in co-culture. Conclusion: B+R combination induced cell cycle arrest and apoptosis in U266 and RPMI-8226. The new drug combination B+R+L has in vitro results comparable with B+L+D and presents an alternative for MM treatment of almost 60% of cases bearing JAK2 overexpression. Our results support future studies using JAK inhibitors as an alternative for MM treatment in a Precision Medicine approach. Financial support: FAPESP 2010/17668-6 and CNPq. Disclosures No relevant conflicts of interest to declare.

Silencing of HSP70 and TRIAP1 Genes in Multiple Myeloma Cell Lines Induces Apoptosis through Apaf-1/Caspase 9 Pathway: New Potential Targets on the Way?

Introduction: Some evidences suggest that heat shock protein 70 (HSP70) is overexpressed in many types of cancer, and that high expression of this chaperone is linked with increasing tumor grade and/or poor prognosis. The overexpression of HSP70 may provide a selective advantage for tumor cell survival, due in part, to its ability in inhibiting cell death via APAF-1 (apoptosis protease activating factor 1) and Caspase 9. The TP53 Regulated Inhibitor of Apoptosis 1 (TRIAP1) gene can modulate apoptotic pathways by interaction with HSP70. Some studies have shown that HSP70 gene silencing using antisense RNA, abundantly induced cell death in breast cancer lines and it was not toxic to normal breast epithelial cells or human fibroblasts. This cell death was not dependent on P53status or inhibited by Bcl2 pathway. Other studies using HSP70 antisense RNA have also indicated apoptosis of tumor cells in lung cancer, oral cavity, colon, prostate, liver, and brain cell lines. Although there are several studies on the role of HSP70 gene in apoptosis and drug resistance, there is a lack of information about this gene in multiple myeloma (MM). Objectives: To analyze the importance of HSP70 and TRIAP1 as potential targets for MM therapy through: 1) stable silencing of HSP70 and TRIAP1 in MM cell lines; 2) evaluation of each gene silencing effect on cell cycle and apoptosis. Methods: The expression of TRIAP1 and HSP70 genes in MM cell lines (U266, SKO-007, SK-MM2 and RPMI8226) was examined by quantitative real time PCR (qPCR). Cell lines were submitted to transduction with pLKO lentiviral vector containing short hairpin RNAs (shRNAs) for silencing the target genes (shRNAHSP70 and shRNATRIAP1). Lentiviral vectors with control sequences (scramble) were used to transduce the same cell lines. Apoptosis was assessed by flow cytometry after annexin V and propidium iodide (PI) staining. We also evaluated APAF-1 and Caspase 9 gene expression by qPCR and Caspase 9 and Caspase 3/7 protein activity. Results: The cell lines RPMI8226 (without deletion of P53 by FISH) and U266 (deletion of one allele of P53 by FISH) were chosen for the transduction experiments because they showed relevant expression of TRIAP1 and HSP70. The efficiency of transduction, as measured in both cell lines transduced with the pLKO vector containing the GFP reporter gene, was 70%, demonstrating that there would be no technical restriction to perform this experiment. RPMI8226 and U266 were submitted to three independent transductions, in triplicate, with the lentiviral vector containing the constructs pLKO shRNATRIAP1, shRNAHSP70 and shRNAscramble. We obtained the silencing of TRIAP1 and HSP70 genes in both MM cell lines when the transduced cell lines were compared with shRNAscramble. Silencing was confirmed by relative qPCR and Western blotting (for HSP70 only). Inhibition of TRIAP1 expression significantly increased the percentage of cells in late apoptosis (annexin V+/Propidium Iodide+) analysis (p<0.001 for RPMI8226 and P<0.01 for U266) one week after transduction and it was accompanied by increased expression of Caspase 9 in both MM cell lines (p<0.001 for RPMI8226 and p<0.05 for U266 cell lines) (One-Way ANOVA with Tukey´s multiple comparison test) when transduced cells were compared with the respective wild type cell lines. Furthermore, the inhibition of TRIAP1 resulted in accumulation of hypodiploid cells after 24 hours of transduction in U266 cell line. Inhibition of HSP70 showed no significant changes in the cell cycle in both MM cell lines. However, we observed an increment in late apoptosis after inhibition of this gene in the two cell lines when the inhibited cells were compared with cells transduced with shRNAscramble one week after transduction (p<0.01 for RPMI8226 and p<0.05 for U266 cell lines) and the results were confirmed by increased activity of Caspase 3/7 (p<0.01 for RPMI8226 and p<0.05 for U266). We observed significantly increase of Caspase 9 activity when RPMI8226 was transduced with shRNAHSP70 (p<0.001). Conclusion: Stable silencing of HSP70 and TRIAP1 in MM cell lines showed a strong impact on the induction of late apoptosis, through APAF-1/Caspase 9 pathway, suggesting that inhibitors of both genes could be exploited as potential targets for the treatment of MM, helping patients whatever P53 status assessed by FISH, as suggested by previous studies in other types of cancer (Support by FAPESP 2010/17668-6). Disclosures No relevant conflicts of interest to declare.

Establishment of Cell Lines from Both Myeloma Bone Marrow and Plasmacytoma: SNU_MM1393_BM and SNU_MM1393_SC from a Single Patient

Purpose.We tried to establish clinically relevant human myeloma cell lines that can contribute to the understanding of multiple myeloma (MM).Materials and Methods.Mononuclear cells obtained from MM patient’s bone marrow were injected via tail vein in an NRG/SCID mouse. Fourteen weeks after the injection, tumor developed at subcutis of the mouse. The engraftment of MM cells into mouse bone marrow (BM) was also observed. We separated and cultured cells from subcutis and BM.Results.After the separation and culture of cells from subcutis and BM, we established two cell lines originating from a single patient (SNU_MM1393_BM and SNU_MM1393_SC). Karyotype of the two newly established MM cell lines showed tetraploidy which is different from the karyotype of the patient (diploidy) indicating clonal evolution. In contrast to SNU_MM1393_BM, cell proliferation of SNU_MM1393_SC was IL-6 independent. SNU_MM1393_BM and SNU_MM1393_SC showed high degree of resistance against bortezomib compared to U266 cell line. SNU_MM1393_BM had the greater lethality compared to SNU_MM1393_SC.Conclusion.Two cell lines harboring different site tropisms established from a single patient showed differences in cytokine response and lethality. Our newly established cell lines could be used as a tool to understand the biology of multiple myeloma.

Functional Studies Of Hsp70 Gene Expression In Multiple Myeloma Cell Lines Suggest That This Gene Inhibition Can Be a Potential Therapeutic Target, Independent Of 17p/p53 Status

Introduction Some evidences show that HSP70 is overexpressed in many cancers, and that high expression of this chaperone correlates with increased tumor grade and/or poor prognosis. Overexpression of HSP70 can provide a selective survival advantage to tumor cells in part due to its ability to inhibit cell death, through APAF-1 and caspase-9 pathway. Currently, some studies have shown that silencing of Hsp70 with antisense RNA induced massive cell death in breast cancer cell lines but was non-toxic to normal breast epithelial cells or normal human fibroblasts. This cell death was not dependent on p53, or inhibited by Bcl2. Other studies indicated that antisense-mediated Hsp70 inhibition in lung, oral, colon, prostate, liver and brain cancer cell lines also causes apoptosis. Despite several studies on the role of Hsp70 gene in apoptosis and drug resistance, there is a lack of information about this gene in multiple myeloma (MM). Objectives To analyze the importance of Hsp70 as a potential target for MM therapy through: 1) Hsp70 stable silencing in MM cell lines; 2) evaluation of the silencing effect of Hsp70 on the cell cycle and apoptosis. Methods MM cell lines (U266, SKO-007, SK-MM2 and RPMI8226) were maintained in RPMI 1640 media supplemented with 10% fetal bovine serum, L-glutamine, MEM NEAA, garamycin, at 37°C in the presence of CO2 atmosphere at 5%. The Hsp70 expression in the four MM cell lines was assessed using real time PCR (qPCR). The relative gene expression was calculated using U266 as control and β-actin as a constitutive gene. Cell lines were submitted to transduction with pLKO lentiviral vector containing the short hairpin RNAs (shRNAs) for Hsp70 silencing (shRNAHsp70). Lentiviral vectors were transduced into the same strains with control sequences (scramble). Apoptosis was analyzed by flow cytometry, using annexin V and propidium iodide (PI), and using APAF-1 and caspase-9 expression by qPCR. Results RPMI8226 and U266 cell lines were chosen for the transduction experiments because their genetic characteristics [the first has normal FISH for 17p and the second has del17p (p53X1)] and good levels of Hsp70 expression. The transduction efficiency, measured in U266 line with pLKO vector with GFP gene, was 70%, demonstrating that there would be no technical restriction to perform this experiment. RPMI8226 and U266 cell lines were subjected to three independent transductions with the pLKO lentiviral vector containing shRNAHsp70 and shRNA scramble, followed by apoptosis evaluation by flow cytometry and qPCR. Hsp70 expression was inhibited in at least 80% of cells in both RPMI8226 and U266 when shRNAHsp70 transduced cell lines were compared with shRNA scramble. Late apoptosis (annexin V+/PI+) analysis, 7 days after transduction, showed statistically significant difference between cell lines transduced with shRNAHsp70 and shRNA scramble (p<0.001 for RPMI8226 and p<0.05 for U266) and between cell lines transduced with shRNAHsp70 and wild type (p<0.0001 for RPMI8226 and p<0.01 for U266) (One-Way ANOVA). We also found a statistically significant difference among relative expression of APAF-1 and caspase-9 in both U266 and RPMI8226 transduced with shRNAHsp70 and wild type or the same cell lines transduced with empty vector, 7 days after transduction, confirming the results obtained at cellular level. Conclusion In line with recent literature, the results obtained here with the Hsp70 stable silencing in MM cell lines RPMI8226 and U266 show a strong impact on the induction of late apoptosis, at gene and cellular levels, through APAF-1/caspase-9 pathway, suggesting that inhibitors of this heat shock can be explored as potential tools in the treatment of MM, using antisense-mediated Hsp70 inhibition, whatever patient's 17p (p53) FISH status (Support by FAPESP 2010/17668-6). Disclosures: No relevant conflicts of interest to declare.