tretinoin and Multiple-Myeloma

Monoclonal antibodies (mAbs) were initially considered as a possible "magic bullet" for in vivo elimination of tumor cells. mAbs represented the first step: however, as they were murine in nature (the earliest experience on the field), they were considered unfit for human applications. This prompted the development of techniques for cloning the variable regions of conventional murine antibodies, genetically mounted on human IgG. The last step in this years-long process was the design for the preparation of fully human reagents. The choice of the target molecule was also problematic, since cancer-specific targets are quite limited in number. To overcome this obstacle in the planning phases of antibody-mediated therapy, attention was focused on a set of normal molecules, whose quantitative distribution may balance a tissue-dependent generalized expression. The results and clinical success obtained with anti-CD20 mAbs revived interest in this type of strategy. Using multiple myeloma (MM) as a tumor model was challenging first of all because the plasma cells and their neoplastic counterpart eluded the efforts of the Workshop on Differentiation Antigens to find a target molecule exclusively expressed by these cells. For this reason, attention was turned to surface molecules which fulfill the requisites of being reasonably good targets, even if not specifically restricted to tumor cells. In 2009, we proposed CD38 as a MM target in virtue of its expression: it is absent on early hematological progenitors, has variable but generalized limited expression by normal cells, but is extremely high in plasma cells and in myeloma. Further, regulation of its expression appeared to be dependent on a variety of factors, including exposure to all-trans retinoic acid (ATRA), a potent and highly specific inducer of CD38 expression in human promyelocytic leukemia cells that are now approved for in vivo use. This review discusses the history of human CD38, from its initial characterization to its targeting in antibody-mediated therapy of human myeloma.

Topics: ADP-ribosyl Cyclase 1; Humans; Membrane Glycoproteins; Multiple Myeloma; Tretinoin

This note mechanistically accounts for recent unexplained findings that all-trans retinoic acid (ATRA, also termed tretinoin) exerts an anti-viral effect against hepatitis C virus (HCV) in chronically infected patients, in whom ATRA also showed synergy with interferon-alpha. How HCV replication was suppressed was unclear. Both effects of ATRA can be accounted for by ATRA's upregulation of RIG protein, an 18 kDa product of retinoic induced gene-1. Increased RIG then couples ATRA to increased Type 1 interferons' production. Details of this mechanism predict that ATRA will similarly augment interferon-a activity in treating chronic myelogenous leukemia, melanoma, myeloma and renal cell carcinoma and that the addition of ribavirin and/or bexarotene will each incrementally enhance interferon-a responses in these cancers.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Bexarotene; Carcinoma, Renal Cell; Drug Screening Assays, Antitumor; Hepacivirus; Humans; Interferon-alpha; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Melanoma; Models, Biological; Multiple Myeloma; Ribavirin; Tetrahydronaphthalenes; Treatment Outcome; Tretinoin

The same progress in the recent therapeutic strategy for older adults with hematological malignancies has also been seen in younger adults. The standard initial therapy for elderly acute promylocytic leukemia is the combination with all-trans retinoic acid and anthracyclines. For other acute myeloid leukemias (AML), many trials of combination chemotherapy have not improved the outcome of elderly patients. Gemtuzumab ozogamicin,which is an immunoconjugate binding to CD 33 on the surface of AML blasts, has produced good results for elderly patients in either monotherapy or in combination with conventional chemotherapeutic drugs. One of the BCR-ABL tyrosine kinase inhibitors, imatinib mesylate, is active for elderly Philadelphia-positive leukemia including acute lymphoblastic leukemia and chronic myeloid leukemia. In the treatment of elderly diffuse large B cell lymphoma, combination of rituximab and cyclophosphamide+doxorubicin+vincristine+prednisone (CHOP) has become the therapy of choice based upon a Groupe d'Etude des Lymphomes de l'Adulte (GELA) trial even though there are some other trials for elderly patients such as dose-dense CHOP therapy. For follicular lymphoma, combination therapies of rituximab and cytotoxic drugs such as R-CHOP and R-CVP are also considered as promising therapies. For the management of multiple myeloma, high-dose chemotherapy, mainly melphalan with autologous stem cell transplantation, has become the standard treatment even for elderly patients less than 65 years of age.

Topics: Aged; Aminoglycosides; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antibodies, Monoclonal, Murine-Derived; Antineoplastic Combined Chemotherapy Protocols; Cyclophosphamide; Doxorubicin; Drug Administration Schedule; Gemtuzumab; Hematologic Neoplasms; Humans; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Leukemia, Myeloid, Acute; Lymphoma, B-Cell; Lymphoma, Large B-Cell, Diffuse; Middle Aged; Multiple Myeloma; Prednisone; Rituximab; Survival Rate; Tretinoin; Vincristine

All-trans retinoic acid (ATRA) is a natural oxidative metabolite of Vitamin A (retinol) and is known to be a regulator of cell proliferation differentiation, especially in various malignant cells. The cyto-differentiating action of ATRA has led to its usage in the treatment of several malignancies, particularly acute promyelocytic leukemia (APL). There have been many reports regarding the cell biological effects of ATRA on human myeloma cells and a few clinical trials. Most of these reports have revealed growth inhibition by ATRA mediated by down-regulation of the IL-6/IL-6R auto/paracrine loop, and upregulation of p21/Cip1. Here, we review previous reports and introduce experimental results obtained using various myeloma cell lines established in our laboratory.

Topics: Antineoplastic Agents; Cell Differentiation; Cell Division; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Humans; Interleukin-6; Multiple Myeloma; Receptors, Interleukin-6; Tretinoin

In addition to interleukin (IL)-6, IL-10 is considered as one of the most important cytokines regulating the proliferation and cellular characteristics of myeloma cells. It is still unclear from the clinical data how serum IL-10 levels of various stages of myeloma, are related to clinical manifestations of this disease. Several studies have reported that IL-10 affects myeloma cells by stimulating secondary signals for cell proliferation through oncostatin M (OSM) and IL-11. In experiments using human myeloma cell lines established at our laboratory, IL-10 seemed to be expressed in half of myelomas simultaneously with OSM, and to be correlated with c-maf, a transcription factor, which has been known to be overexpressed in myelomas with t(14;16)(q32;q23). In addition, IL-10 abolishes all trans retinoic acid (ATRA)-induced growth inhibition of myeloma cells. The expression and production of IL-10 in myeloma patients may be important for sub-categorization and the establishment of a case-oriented therapy.

Topics: Cell Division; Cytokines; Humans; Interleukin-10; Multiple Myeloma; Tretinoin

Acute promyelocytic leukemia (APL), once considered the most devastating subtype of acute myeloid leukemia, is now the most treatable of all subtypes as a result of intensive research into its molecular pathogenesis. This research has led to a rational approach to treatment in which the use of the differentiating agent all-trans-retinoic acid (ATRA) has proven to be effective first-line treatment for inducing complete remission. Arsenic trioxide (ATO) is currently used to treat relapsed disease, further enhancing survival rates in a patient population for which limited salvage options exist. This review discusses the molecular mechanisms responsible for development of APL and the evolution of treatment options over the last three decades, including the major advances using ATRA and ATO in the last 12 years. The mechanism of action of ATO is also described in view of this agent's potential for broader therapeutic application in a variety of hematologic malignancies.

Topics: Antineoplastic Agents; Arsenic Trioxide; Arsenicals; Clinical Trials as Topic; Hematologic Neoplasms; Humans; Leukemia, Promyelocytic, Acute; Multiple Myeloma; Myelodysplastic Syndromes; Oxides; Recurrence; Remission Induction; Survival Analysis; Treatment Outcome; Tretinoin

Topics: Activins; Animals; Apoptosis; Humans; Inhibins; Multiple Myeloma; Tretinoin

The clinical pharmacology of all-trans retinoic acid (RA) has distinct differences from that of its widely studied stereoisomer 13-cis retinoic acid (cRA). RA is much more rapidly cleared from plasma following oral administration; their respective half-lives are < 1 h and 13 h. There is extensive accumulation of the 4-oxo-cRA in plasma following repeated doses of cRA, while 4-oxo-RA is only a minor metabolite in plasma following RA administration. The extent of isomerization in vivo differs for the two retinoids. In contrast to cRA, where up to a 1:3 ratio of RA to cRA is observed in patient plasma following drug administration, cRA concentrations in excess of 10 ng/ml are rarely observed in plasma of patients receiving exogenous RA. RA administration produces autoinduction of its own oxidative catabolism; this effect does not occur with cRA. These pharmacokinetic differences have been observed in leukemia and solid tumor patients. Detailed analysis of the results of the population studied suggest that both constitutive and RA-induced hypercatabolism of RA occurs. Both of these hypercatabolic states can be modulated by concurrent administration of ketoconazole, an inhibitor of cytochrome P-450 and lipoxygenase-mediated oxidations.

Topics: Carcinoma, Non-Small-Cell Lung; Humans; Ketoconazole; Leukemia, Promyelocytic, Acute; Lung Neoplasms; Male; Metabolic Clearance Rate; Multiple Myeloma; Oxidation-Reduction; Prostatic Neoplasms; Tretinoin

The efficacy of daratumumab depends partially on CD38 expression on multiple myeloma (MM) cells. We have previously shown that all-trans retinoic acid (ATRA) upregulates CD38 expression and reverts daratumumab-resistance ex vivo. We therefore evaluated the optimal dose, efficacy, and safety of daratumumab combined with ATRA in patients with daratumumab-refractory MM in a phase 1/2 study (NCT02751255). In part A of the study, 63 patients were treated with daratumumab monotherapy. Fifty patients with daratumumab-refractory MM were subsequently enrolled in part B and treated with daratumumab (reintensified schedule) combined with ATRA until disease progression. The recommended phase 2 dose of ATRA in combination with daratumumab was defined as 45 mg/m2. At this dose, the overall response rate (ORR) was 5%, indicating that the primary endpoint (ORR ≥15%) was not met. However, most patients (66%) achieved at least stable disease. After a median follow-up of 43 months, the median progression-free survival (PFS) for all patients was 2.8 months. Patients who previously achieved at least a partial response or minimal response/stable disease with prior daratumumab monotherapy had a significantly longer PFS compared with patients who immediately progressed during daratumumab as single agent (median PFS 3.4 and 2.8 vs 1.3 months). The median overall survival was 19.1 months. The addition of ATRA did not increase the incidence of adverse events. Flow cytometric analysis revealed that ATRA temporarily increased CD38 expression on immune cell subsets. In conclusion, the addition of ATRA and reintensification of daratumumab had limited activity in patients with daratumumab-refractory MM, which may be explained by the transient upregulation of CD38 expression. This trial was registered at www.clinicaltrials.gov as #NCT02751255.

Topics: Antibodies, Monoclonal; Antineoplastic Combined Chemotherapy Protocols; Humans; Multiple Myeloma; Tretinoin

The anti-CD38 monoclonal antibody daratumumab is well tolerated and has high single agent activity in heavily pretreated relapsed and refractory multiple myeloma (MM). However, not all patients respond, and many patients eventually develop progressive disease to daratumumab monotherapy. We therefore examined whether pretreatment expression levels of CD38 and complement-inhibitory proteins (CIPs) are associated with response and whether changes in expression of these proteins contribute to development of resistance. In a cohort of 102 patients treated with daratumumab monotherapy (16 mg/kg), we found that pretreatment levels of CD38 expression on MM cells were significantly higher in patients who achieved at least partial response (PR) compared with patients who achieved less than PR. However, cell surface expression of the CIPs, CD46, CD55, and CD59, was not associated with clinical response. In addition, CD38 expression was reduced in both bone marrow-localized and circulating MM cells, following the first daratumumab infusion. CD38 expression levels on MM cells increased again following daratumumab discontinuation. In contrast, CD55 and CD59 levels were significantly increased on MM cells only at the time of progression. All-trans retinoic acid increased CD38 levels and decreased CD55 and CD59 expression on MM cells from patients who developed daratumumab resistance, to approximately pretreatment values. This resulted in significant enhancement of daratumumab-mediated complement-dependent cytotoxicity. Together, these data demonstrate an important role for CD38 and CIP expression levels in daratumumab sensitivity and suggest that therapeutic combinations that alter CD38 and CIP expression levels should be investigated in the treatment of MM. These trials were registered at www.clinicaltrials.gov as #NCT00574288 (GEN501) and #NCT01985126 (SIRIUS).

Topics: ADP-ribosyl Cyclase 1; Antibodies, Monoclonal; CD55 Antigens; CD59 Antigens; Clone Cells; Complement Inactivating Agents; Cytotoxicity, Immunologic; Disease Progression; Drug Resistance, Neoplasm; Humans; Multiple Myeloma; Tretinoin

Topics: Antigens, CD34; Blood Platelets; Female; Granulocyte Colony-Stimulating Factor; Hematopoietic Stem Cell Transplantation; Humans; Lymphoma, Non-Hodgkin; Male; Multiple Myeloma; Pilot Projects; Platelet Count; Transplantation Conditioning; Tretinoin

High dose chemotherapy with supporting autologous stem cell transplantation is now considered the treatment of choice in patients with multiple myeloma <65 years old. The best regimen appears to be VAD (vincristine, doxorubicin and dexamethasone), but acute and late toxicity can limit the use of this combination. The use of biological modifiers has not been considered in this situation. We developed a new cytoreductive regimen, in an attempt to retain clinical efficacy but reduce toxicity.. Thirty-six patients, previously untreated with diagnosis of multiple myeloma were enrolled to received the DAI regimen (dexamethasone 30 mg/m(2), i.v., days 1-4, all-trans-retinoic acid 45 mg/m(2), p.o., days 5-14 and interferon alpha 2a, 4.5 MU s.c., days 5-14) administered every 28 days for six cycles before high-dose chemotherapy (melphalan 200 g/m(2)) and autologous stem cell transplantation.. Overall response was observed in 29 cases (80%), complete response in 19 and partial response in 10 patients. Five patients were >65 years old and were treated with dexamethasone/thalidomide. Twenty-four patients underwent transplants. At a median follow-up of 31.6 months, no relapse or disease progression was observed, thus actuarial curves at 3-years showed that event-free survival was 86% and overall survival was 94%. Toxicity was mild.. This regimen appears to be an excellent alternative as cytoreductive treatment before high-dose chemotherapy and autologous stem cell transplantation with excellent overall response and minimal toxicity. Controlled clinical trials are warranted to define the role of this new therapeutic approach.

Topics: Administration, Oral; Adult; Antineoplastic Agents, Alkylating; Antineoplastic Combined Chemotherapy Protocols; Dexamethasone; Disease Progression; Disease-Free Survival; Female; Humans; Immunologic Factors; Infusions, Intravenous; Interferon alpha-2; Interferon-alpha; Male; Melphalan; Middle Aged; Multiple Myeloma; Peripheral Blood Stem Cell Transplantation; Recombinant Proteins; Transplantation, Autologous; Treatment Outcome; Tretinoin

Treatment in patients with multiple myeloma remain to be defined. Younger patients (defined as a cut-off level < 65 years old) will be treated with chemotherapy and transplant procedures. However, most patients > 65 years old are not candidates for this therapeutic approach and the use of intensive chemotherapy could be associated to severe toxicity. We developed an new, not-cytotoxic regimen with dexamethasone 30 mg/m(2), iv, days 1 to 4, all trans retinoic acid 45 mg/m(2), po, days 5 to 14 and interferon alfa 2a 4.5 MU, sc, daily, days 5 to 14 (DAI regimen) administered every 28 days in number of 6 cycles, at this point patients were restaging, if they showed complete response, objective response or partial response they were conducted to received thalidomide 100-200 mg po, daily and dexamethasone 10 mg/2, po days 1 to 4 at monthly intervals, for 18 months. Forty one patients were enrolled in an Phase II study. In an intent to treat analysis all patients were evaluable. Complete response was observed in 18 cases (43%), objective response in 10 patients (24%) and partial response in 5 patients (12%), overall response rate was 80%. Eight patients were considered failures. At an median of 36 months, no relapse of progression disease has been observed, thus actuarial curves at 3-years showed that event free survival is 100% and overall survival is 91%. Toxicity was mild, all patients received the planned dose in time. This regimen appear to be useful in older patients with multiple myeloma, the response rate is higher and toxicity was mild. Controlled clinical trials comparing with conventional chemotherapy will be conducted to define the role of this therapeutic approach.

Topics: Aged; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Dexamethasone; Disease-Free Survival; Female; Humans; Immunosuppressive Agents; Interferon alpha-2; Interferon-alpha; Male; Multiple Myeloma; Recombinant Proteins; Thalidomide; Tretinoin

All-trans retinoic acid (ATRA) is a derivative of vitamin A. ATRA inhibits the growth of human myeloma cell lines and freshly isolated myeloma cells in vitro mainly by down-regulating interleukin-6 receptor. Clinically, however, ATRA alone has not been efficacious and adverse events, notably hypercalcemia, have been common. In the present study 10 patients with stable multiple myeloma after conventional chemotherapy received ATRA alone for 2 months, followed by a combination of ATRA and the chemotherapy regimen during which no further reduction of the paraprotein had occurred. The purpose of the combination therapy was to sensitize the myeloma cells with ATRA to chemotherapy by blocking the growth-promoting effect of IL-6. Although ATRA was well tolerated, ATRA alone lacked clinical efficacy. The combination therapy resulted minimal responses in 4 patients and relatively long progression-free survival in 4 patients was achieved. In 3 of these responding patients serum concentrations of interleukin-6 and/or soluble interleukin-6 receptor were elevated prior to the study. The bone marrow cells of responding patients were sensitive to ATRA in vitro. These results show that ATRA alone is not effective to treat multiple myeloma. There may be some beneficial effect of ATRA in combination chemotherapy in selected patients who have activated IL-6 signaling.

Topics: Aged; Antineoplastic Combined Chemotherapy Protocols; Bone Marrow Cells; Cell Division; Female; Humans; Interleukin-6; Male; Middle Aged; Multiple Myeloma; Oncostatin M; Peptides; Receptors, Interleukin-6; Treatment Outcome; Tretinoin

Topics: Adult; Aged; Antineoplastic Combined Chemotherapy Protocols; Female; Humans; Interferon-alpha; Male; Middle Aged; Multiple Myeloma; Salvage Therapy; Treatment Failure; Tretinoin

Few effective regimen are available for patients with refractory multiple myeloma (RMM). Generally, responses are scarce and disease free survival is very short. We developed a new therapeutic option in these patients using dexamethasone (40 mg/m2, i.v., daily, days 1 to 4), all-trans retinoic acid (45 mg/m2, po, daily, days 5 to 14) and interferon alpha 2a (9.0 MU, daily, subcutaneously, days 5 to 14). The treatment was administered every 21 days for 6 cycles. In a pilot study, 12 patients, heavily treated with chemotherapy and radiotherapy and in some cases with interferon, were allocated to receive the afore mentioned treatment. Response was observed in 10 patients (83%). With a median follow-up of 36.1 months (range 27 to 41), seven patients remain alive and disease-free without any treatment. Two patients were failures and have died due to tumor progression. Toxicity was mild and all patients received treatment according to the planned doses of drugs. The use of biological modifiers in combination with dexamethasone offer a safe and effective therapeutic option in patients with refractory multiple myeloma. More studies are warranted to define the role of this type of treatment.

Topics: Adult; Aged; Antineoplastic Agents, Hormonal; Combined Modality Therapy; Dexamethasone; Disease-Free Survival; Female; Follow-Up Studies; Humans; Hyperglycemia; Immunologic Factors; Interferon alpha-2; Interferon-alpha; Male; Middle Aged; Multiple Myeloma; Pilot Projects; Recombinant Proteins; Treatment Outcome; Tretinoin

All-trans retinoic acid (ATRA) inhibits human myeloma cell growth in vitro, presumably through the down-regulation of interleukin 6 receptors (IL-6R). Based on these and other studies, we initiated a phase II clinical trial using ATRA in patients with advanced refractory multiple myeloma (MM). We report that three out of six treated patients developed severe hypercalcaemia following administration of ATRA, which was accompanied by a significant rise in serum IL-6 levels. Normal calcium levels were restored after the discontinuation of the drug and the administration of standard anti-hypercalcaemic care. We suspect that down-regulation of IL-6R resulted in increased serum IL-6 levels, leading to advanced bone resorption and hypercalcaemia. We conclude that the use of ATRA in patients with advanced MM is not warranted.

Topics: Humans; Hypercalcemia; Interleukin-6; Multiple Myeloma; Tretinoin

B-cell maturation antigen (BCMA) is the lead antigen for chimeric antigen receptor (CAR) T-cell therapy in multiple myeloma (MM). A challenge is inter- and intra-patient heterogeneity in BCMA expression on MM cells and BCMA downmodulation under therapeutic pressure. Accordingly, there is a desire to augment and sustain BCMA expression on MM cells in patients that receive BCMA-CAR T-cell therapy. We used all-trans retinoic acid (ATRA) to augment BCMA expression on MM cells and to increase the efficacy of BCMA-CAR T cells in pre-clinical models. We show that ATRA treatment leads to an increase in BCMA transcripts by quantitative reverse transcription polymerase chain reaction and an increase in BCMA protein expression by flow cytometry in MM cell lines and primary MM cells. Analyses with super-resolution microscopy confirmed increased BCMA protein expression and revealed an even distribution of non-clustered BCMA molecules on the MM cell membrane after ATRA treatment. The enhanced BCMA expression on MM cells after ATRA treatment led to enhanced cytolysis, cytokine secretion and proliferation of BCMA-CAR T cells in vitro, and increased efficacy of BCMA-CAR T-cell therapy in a murine xenograft model of MM in vivo (NSG/MM.1S). Combination treatment of MM cells with ATRA and the γ- secretase inhibitor crenigacestat further enhanced BCMA expression and the efficacy of BCMA-CAR T-cell therapy in vitro and in vivo. Taken together, the data show that ATRA treatment leads to enhanced BCMA expression on MM cells and consecutively, enhanced reactivity of BCMA-CAR T cells. The data support the clinical evaluation of ATRA in combination with BCMA-CAR T-cell therapy and potentially, other BCMA-directed immunotherapies.

Topics: Amyloid Precursor Protein Secretases; Animals; B-Cell Maturation Antigen; Humans; Immunotherapy, Adoptive; Mice; Multiple Myeloma; Receptors, Chimeric Antigen; T-Lymphocytes; Tretinoin

Immunotherapies targeting CD38 have demonstrated salient efficacy in relapsed/refractory multiple myeloma (MM). However, loss of CD38 antigen and outgrowth of CD38 negative plasma cells have emerged as a major obstacle in clinics. All-trans retinoic acid (ATRA) has been reported to upregulate CD38 expression, but the mechanism and adaptive genetic background remain unexplored.. We report that ATRA upregulates MM cells CD38 in a non-linear manner, which is t(4;14) translocation dependent, and t(4;14) translocation-induced NSD2 shows positive correlation with ATRA-induced level of, but not with basal level of CD38 expression. Mechanistically, NSD2 interacts with the ATRA receptor, RARα, and protects it from degradation. Meanwhile, NSD2 enhances the nuclear condensation of RARα and modifies the histone H3 dimethylation at lysine 36 on CD38 promoter. Knockdown of NSD2 attenuates the sensitization of MM against ATRA induced CD38 upregulation. Translationally, ATRA is prone to augment the efficacy of anti-CD38 CAR T cells in NSD2. This study elucidates a mechanism of ATRA in regulating CD38 expression and expands the clinical potential of ATRA in improving immunotherapies against CD38 in patients with MM.Cite Now.

Topics: Animals; Immunotherapy, Adoptive; Mice; Mice, Inbred NOD; Multiple Myeloma; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Tretinoin

CD38 expression on myeloma cells is a critical factor affecting the early response to the anti-CD38 antibody daratumumab. However, factors affecting CD38 expression in untreated multiple myeloma are not fully elucidated. In this study, we found that CD38 expression was significantly lower in myeloma patients with the translocation t(11;14)-associated immature plasma cell phenotype, and particularly in those expressing B-cell-associated genes such as PAX5 and CD79A. CD138, a representative marker of plasmacytic differentiation, was also significantly lower in these patients, suggesting that CD38 expression may be associated with the differentiation and maturation stages of myeloma cells. Furthermore, the BCL2/BCL2L1 ratio, a response marker of the BCL2 inhibitor venetoclax, was significantly higher in patients with the immature phenotype expressing B-cell-associated genes. The BCL2/BCL2L1 ratio and CD38 expression were significantly negatively correlated. We also confirmed that patients with translocation t(11;14) expressing B-cell-associated genes were indeed less sensitive to daratumumab-mediated direct cytotoxicity but highly sensitive to venetoclax treatment in ex vivo assays. Moreover, all-trans-retinoic acid, which enhances CD38 expression and induces cell differentiation in myeloma cells, reduced B-cell marker expression and the BCL2/BCL2L1 ratio in myeloma cell lines, leading to reduced efficacy of venetoclax. Venetoclax specifically induces cell death in myeloma with t(11;14), although why patients with translocation t(11;14) show BCL2 dependence is unclear. These results suggest that BCL2 dependence, as well as CD38 expression, are deeply associated with the differentiation and maturation stages of myeloma cells. This study highlights the importance of examining t(11;14) and considering cell maturity in myeloma treatment strategies.

Topics: ADP-ribosyl Cyclase 1; Adult; Aged; Aged, 80 and over; Antibodies, Monoclonal; Antineoplastic Agents; B-Lymphocytes; Bridged Bicyclo Compounds, Heterocyclic; Cell Differentiation; Cell Line, Tumor; Chromosomes, Human, Pair 11; Chromosomes, Human, Pair 14; Female; Humans; Male; Membrane Glycoproteins; Middle Aged; Multiple Myeloma; Phenotype; Proto-Oncogene Proteins c-bcl-2; Signal Transduction; Sulfonamides; Translocation, Genetic; Tretinoin

Multiple myeloma (MM) is a plasma cell neoplasm that commonly expresses CD38. Daratumumab (DARA), a human monoclonal antibody targeting CD38, has significantly improved the outcome of patients with relapsed or refractory MM, but the response is transient in most cases. Putative mechanisms of suboptimal efficacy of DARA include downregulation of CD38 expression and overexpression of complement inhibitory proteins on MM target cells as well as DARA-induced depletion of CD38high natural killer (NK) cells resulting in crippled antibody-dependent cellular cytotoxicity (ADCC). Here, we tested whether maintaining NK cell function during DARA therapy could maximize DARA-mediated ADCC against MM cells and deepen the response. We used the CRISPR/Cas9 system to delete CD38 (CD38KO) in ex vivo expanded peripheral blood NK cells. These CD38KO NK cells were completely resistant to DARA-induced fratricide, showed superior persistence in immune-deficient mice pretreated with DARA, and enhanced ADCC activity against CD38-expressing MM cell lines and primary MM cells. In addition, transcriptomic and cellular metabolic analysis demonstrated that CD38KO NK cells have unique metabolic reprogramming with higher mitochondrial respiratory capacity. Finally, we evaluated the impact of exposure to all-trans retinoic acid (ATRA) on wild-type NK and CD38KO NK cell function and highlighted potential benefits and drawbacks of combining ATRA with DARA in patients with MM. Taken together, these findings provide proof of concept that adoptive immunotherapy using ex vivo expanded CD38KO NK cells has the potential to boost DARA activity in MM.

Topics: Adoptive Transfer; ADP-ribosyl Cyclase 1; Animals; Antibodies, Monoclonal; Antibody-Dependent Cell Cytotoxicity; Cell Line, Tumor; CRISPR-Cas Systems; Cytotoxicity, Immunologic; Humans; Immunotherapy; Killer Cells, Natural; Male; Membrane Glycoproteins; Mice; Mice, Inbred NOD; Multiple Myeloma; NAD; Oxidative Phosphorylation; Specific Pathogen-Free Organisms; Tretinoin; Whole Genome Sequencing

Topics: ADP-ribosyl Cyclase 1; Antineoplastic Combined Chemotherapy Protocols; Histone Deacetylase 6; Histone Deacetylase Inhibitors; Humans; Interferon-alpha; Membrane Glycoproteins; Multiple Myeloma; Tretinoin

This article considers the problem of designing Phase I-II clinical trials with delayed toxicity and efficacy outcomes. The proposed design is motivated by a Phase I-II study evaluating all-trans retinoic acid (ATRA) in combination with a fixed dose of daratumumab in the treatment of relapsed or refractory multiple myeloma. The primary objective of the study is to identify a dose that maximizes efficacy and has an acceptable level of toxicity. The toxicity endpoint is observed in one cycle of therapy (

Topics: Antibodies, Monoclonal; Clinical Trials, Phase I as Topic; Clinical Trials, Phase II as Topic; Humans; Multiple Myeloma; Research Design; Tretinoin

Receptor-targeted delivery of imaging and therapeutic agents has emerged as an attractive strategy to diagnosis and treat many diseases including cancer. One of the most well-studied receptors for targeted therapies is the folate receptor (FR) family. FR-α and FR-β are present on many cancers with little expression in normal tissues; leading to the testing of at least six folate-targeted drugs in human clinical trials for various cancers. However, the expression of FR in blood cancers has not been fully explored with no reports of FR expression in myelomas. Herein, we report the expression of both FR-α and FR-β on CD138 + plasma cells isolated from patients with multiple myeloma. In addition, all-trans retinoic acid was shown to increase the levels of FR-α and FR-β in two myeloma cell lines. Altogether, this data suggests that folate-targeted therapies for the treatment of multiple myeloma warrants further investigation.

Topics: Antineoplastic Agents; Bone Marrow; Cell Line, Tumor; Folate Receptor 1; Folate Receptor 2; Gene Expression Regulation; Humans; Multiple Myeloma; Plasma Cells; Syndecan-1; Tretinoin; Up-Regulation

The significance of quantitative changes of ALDH1A1 and RDH10 gene expression in 22 non-treated multiple myeloma patients were studied. We found a direct correlation between the expression of ALDH1A1 and RDH10 genes. We showed that ALDHA1 and RDH10 expression were inversely related with expression of a key gene for all-trans-retinoic acid catabolism, CYP26A1, and correlated with expression of RARα and PPARβ/ genes. In addition for the first time it was re- vealed that increased expression of ALDH1A1-RDH10-RARα- PPARβ/δ pattern could be considered as adverse prognostic factor associated with a higher concentration of paraprotein and worst overall survival of patients with newly diagnosed multiple myeloma.

Topics: Adult; Aged; Aged, 80 and over; Alcohol Oxidoreductases; Aldehyde Dehydrogenase; Aldehyde Dehydrogenase 1 Family; Female; Gene Expression Regulation, Neoplastic; Humans; Male; Middle Aged; Multiple Myeloma; Neoplasm Proteins; PPAR delta; PPAR-beta; Retinal Dehydrogenase; Retinoic Acid Receptor alpha; Tretinoin

Interactions between multiple myeloma (MM) cells and the BM microenvironment play a critical role in bortezomib (BTZ) resistance. However, the mechanisms involved in these interactions are not completely understood. We previously showed that expression of CYP26 in BM stromal cells maintains a retinoic acid-low (RA-low) microenvironment that prevents the differentiation of normal and malignant hematopoietic cells. Since a low secretory B cell phenotype is associated with BTZ resistance in MM and retinoid signaling promotes plasma cell differentiation and Ig production, we investigated whether stromal expression of the cytochrome P450 monooxygenase CYP26 modulates BTZ sensitivity in the BM niche. CYP26-mediated inactivation of RA within the BM microenvironment prevented plasma cell differentiation and promoted a B cell-like, BTZ-resistant phenotype in human MM cells that were cocultured on BM stroma. Moreover, paracrine Hedgehog secretion by MM cells upregulated stromal CYP26 and further reinforced a protective microenvironment. These results suggest that crosstalk between Hedgehog and retinoid signaling modulates BTZ sensitivity in the BM niche. Targeting these pathological interactions holds promise for eliminating minimal residual disease in MM.

Topics: Bortezomib; Cell Differentiation; Cell Line, Tumor; Coculture Techniques; Cytochrome P450 Family 26; Drug Resistance, Neoplasm; Hedgehog Proteins; Humans; Multiple Myeloma; Neoplasm Proteins; Paracrine Communication; Plasma Cells; Stromal Cells; Tretinoin; Tumor Microenvironment

Daratumumab is an anti-CD38 monoclonal antibody with lytic activity against multiple myeloma (MM) cells, including ADCC (antibody-dependent cellular cytotoxicity) and CDC (complement-dependent cytotoxicity). Owing to a marked heterogeneity of response to daratumumab therapy in MM, we investigated determinants of the sensitivity of MM cells toward daratumumab-mediated ADCC and CDC. In bone marrow samples from 144 MM patients, we observed no difference in daratumumab-mediated lysis between newly diagnosed or relapsed/refractory patients. However, we discovered, next to an expected effect of effector (natural killer cells/monocytes) to target (MM cells) ratio on ADCC, a significant association between CD38 expression and daratumumab-mediated ADCC (127 patients), as well as CDC (56 patients). Similarly, experiments with isogenic MM cell lines expressing different levels of CD38 revealed that the level of CD38 expression is an important determinant of daratumumab-mediated ADCC and CDC. Importantly, all-trans retinoic acid (ATRA) increased CD38 expression levels but also reduced expression of the complement-inhibitory proteins CD55 and CD59 in both cell lines and primary MM samples. This resulted in a significant enhancement of the activity of daratumumab in vitro and in a humanized MM mouse model as well. Our results provide the preclinical rationale for further evaluation of daratumumab combined with ATRA in MM patients.

Topics: ADP-ribosyl Cyclase 1; Adult; Aged; Aged, 80 and over; Animals; Antibodies, Monoclonal; Antibody-Dependent Cell Cytotoxicity; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Bone Marrow; Cell Proliferation; Cytotoxicity, Immunologic; Disease Models, Animal; DNA-Binding Proteins; Drug Resistance, Neoplasm; Drug Synergism; Female; Flow Cytometry; Humans; Male; Membrane Glycoproteins; Mice; Middle Aged; Multiple Myeloma; Neoplasm Recurrence, Local; Salvage Therapy; Tretinoin; Tumor Cells, Cultured

Resistance to the proteasome inhibitor bortezomib is an emerging clinical problem whose mechanisms have not been fully elucidated. We considered the possibility that this could be associated with enhanced proteasome activity in part through the action of the proteasome maturation protein (POMP). Bortezomib-resistant myeloma models were used to examine the correlation between POMP expression and bortezomib sensitivity. POMP expression was then modulated using genetic and pharmacologic approaches to determine the effects on proteasome inhibitor sensitivity in cell lines and in vivo models. Resistant cell lines were found to overexpress POMP, and while its suppression in cell lines enhanced bortezomib sensitivity, POMP overexpression in drug-naive cells conferred resistance. Overexpression of POMP was associated with increased levels of nuclear factor (erythroid-derived 2)-like (NRF2), and NRF2 was found to bind to and activate the POMP promoter. Knockdown of NRF2 in bortezomib-resistant cells reduced POMP levels and proteasome activity, whereas its overexpression in drug-naive cells increased POMP and proteasome activity. The NRF2 inhibitor all-trans-retinoic acid reduced cellular NRF2 levels and increased the anti-proliferative and pro-apoptotic activities of bortezomib in resistant cells, while decreasing proteasome capacity. Finally, the combination of all-trans-retinoic acid with bortezomib showed enhanced activity against primary patient samples and in a murine model of bortezomib-resistant myeloma. Taken together, these studies validate a role for the NRF2/POMP axis in bortezomib resistance and identify NRF2 and POMP as potentially attractive targets for chemosensitization to this proteasome inhibitor.

Topics: Bortezomib; Cell Line, Tumor; Drug Resistance, Neoplasm; Humans; Molecular Chaperones; Multiple Myeloma; NF-E2-Related Factor 2; Tretinoin

Resistance of myeloma to lenalidomide is an emerging clinical problem, and though it has been associated in part with activation of Wnt/β-catenin signaling, the mediators of this phenotype remained undefined. Lenalidomide-resistant models were found to overexpress the hyaluronan (HA)-binding protein CD44, a downstream Wnt/β-catenin transcriptional target. Consistent with a role of CD44 in cell adhesion-mediated drug resistance (CAM-DR), lenalidomide-resistant myeloma cells were more adhesive to bone marrow stroma and HA-coated plates. Blockade of CD44 with monoclonal antibodies, free HA or CD44 knockdown reduced adhesion and sensitized to lenalidomide. Wnt/β-catenin suppression by FH535 enhanced the activity of lenalidomide, as did interleukin-6 neutralization with siltuximab. Notably, all-trans retinoic acid (ATRA) downregulated total β-catenin, cell-surface and total CD44, reduced adhesion of lenalidomide-resistant myeloma cells and enhanced the activity of lenalidomide in a lenalidomide-resistant in vivo murine xenograft model. Finally, ATRA sensitized primary myeloma samples from patients that had relapsed and/or refractory disease after lenalidomide therapy to this immunomodulatory agent ex vivo. Taken together, our findings support the hypotheses that CD44 and CAM-DR contribute to lenalidomide resistance in multiple myeloma, that CD44 should be evaluated as a putative biomarker of sensitivity to lenalidomide, and that ATRA or other approaches that target CD44 may overcome clinical lenalidomide resistance.

Topics: Animals; Antibodies, Monoclonal; Antibody-Dependent Cell Cytotoxicity; Antineoplastic Agents; Cell Adhesion; Cell Line, Tumor; Disease Models, Animal; Drug Resistance, Neoplasm; Gene Expression; Humans; Hyaluronan Receptors; Hyaluronic Acid; Immunologic Factors; Lenalidomide; Mice; Multiple Myeloma; Protein Binding; Thalidomide; Tretinoin; Xenograft Model Antitumor Assays

Despite the successful application of all-trans retinoic acid (ATRA) in multiple myeloma treatment, ATRA-induced chemoresistance in the myeloma patients is very common in clinic. In this study, we evaluated the effect of ATRA on the expression of apurinic endonuclease/redox factor-1 (Ape/Ref-1) in the U266 and RPMI-8226 myeloma cells to explore the chemoresistance mechanism involved. ATRA treatment induced upregulation of Ape/Ref-1 via a noncanonical signaling pathway, leading to enhanced pro-survival activity counteracting melphalan (an alkylating agent). ATRA rapidly activated p38-MSK (mitogen- and stress activated protein kinase) cascade to phosphorylate cAMP response element-binding protein (CREB). Phosphorylated CREB was recruited to the Ape/Ref-1 promoter to evoke the gene expression. The stimulation of ATRA on Ape/Ref-1 expression was attenuated by either p38-MSK inhibitors or overexpression of dominant-negative MSK1 mutants. Moreover, ATRA-mediated Ape/Ref-1 upregulation was correlated with histone modification and activation of CBP/p300, an important cofactors for CREB transcriptional activity. C646, a competitive CBP/p300 inhibitor, abolished the upregulation of Ape/Ref-1 induced by ATRA. Intriguingly, CBP rather than p300 played a dominant role in the expression of Ape/Ref-1. Hence, our study suggests the existence of a noncanonical mechanism involving p38-MSK-CREB cascade and CBP induction to mediate ATRA-induced Ape/Ref-1 expression and acquired chemoresistance in myeloma cells.

Topics: Apoptosis; Cell Death; Cell Line, Tumor; Cell Proliferation; Cyclic AMP Response Element-Binding Protein; DNA-(Apurinic or Apyrimidinic Site) Lyase; Drug Resistance, Neoplasm; Gene Expression Regulation, Neoplastic; Humans; Melphalan; Multiple Myeloma; p300-CBP Transcription Factors; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Ribosomal Protein S6 Kinases, 90-kDa; Signal Transduction; Tretinoin; Up-Regulation

To investigate the effect of all-trans retinoid acid (ATRA) and granulocyte colony-stimulating factor (G-CSF) on the growth, apoptosis, differentiation and expression of RARα2 of myeloma cells.. Myeloma cell lines OPM2 (RARα2 positive) and U266 (RARα2 negative) were treated with ATRA in the presence or absence of G-CSF. The cells were divided into 6 groups: control groups, G-CSF groups (treated with 1000 U/ml and 2000 U/ml), ATRA groups (treated with 1.0 μmol/L ATRA) and combined groups (treated with 1000 U/mL or 2000 U/mL G-CSF plus 1.0 μmol/L ATRA). The cell viability, growth and apoptosis were examined by MTT method, inverted microscopy and Annexin-V/PI staining, respectively; RARα2 expression was detected by reverse transcription PCR; morphology change was evaluated by Wright-Giemsa staining; CD49e expression were analyzed by flow cytometry.. The proliferation of OPM2 cells was inhibited by ATRA treatment (P<0.05) . The growth inhibition rates in combined groups were higher than corresponding single ATRA groups (P<0.05). However, the above effects in U266 cells were not significant (P >0.05). The OPM2 cell stained by Wright-Giemsa in ATRA groups showed that the cell nucleus became smaller, chromatin condensed, number of nucleolus reduced, the volume of cytoplasm increased and the cytoplasm became dark blue. Expression rates of CD49e were low in both U266 and OPM2 cells. Expression of RARα2 in OPM2 cells of combination groups were higher than those of control group and corresponding single groups (P<0.05); and there was no significant difference between control group and G-CSF groups (P>0.05). Expression of RARα2 in U266 cells of control group and G-CSF groups was not detected; and ATRA groups and combination groups had weak expression.. ATRA can induce proliferation inhibition in RARα2-expressing myeloma cells, and it may also play a certain role in promoting differentiation of RARα2 positive myeloma cells.

Topics: Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Granulocyte Colony-Stimulating Factor; Humans; Multiple Myeloma; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Tretinoin

The deregulation of B cell differentiation has been shown to contribute to autoimmune disorders, hematological cancers, and aging. We provide evidence that the retinoic acid-producing enzyme aldehyde dehydrogenase 1a1 (Aldh1a1) is an oncogene suppressor in specific splenic IgG1(+)/CD19(-) and IgG1(+)/CD19(+) B cell populations. Aldh1a1 regulated transcription factors during B cell differentiation in a sequential manner: 1) retinoic acid receptor alpha (Rara) in IgG1(+)/CD19(-) and 2) zinc finger protein Zfp423 and peroxisome proliferator-activated receptor gamma (Pparg) in IgG1(+)/CD19(+) splenocytes. In Aldh1a1(-/-) mice, splenic IgG1(+)/CD19(-) and IgG1(+)/CD19(+) B cells acquired expression of proto-oncogenic genes c-Fos, c-Jun, and Hoxa10 that resulted in splenomegaly. Human multiple myeloma B cell lines also lack Aldh1a1 expression; however, ectopic Aldh1a1 expression rescued Rara and Znf423 expressions in these cells. Our data highlight a mechanism by which an enzyme involved in vitamin A metabolism can improve B cell resistance to oncogenesis.

Topics: Aldehyde Dehydrogenase; Aldehyde Dehydrogenase 1 Family; Animals; Antigens, CD19; B-Lymphocytes; Cell Differentiation; Cell Line, Tumor; DNA-Binding Proteins; Female; Gene Expression Regulation, Neoplastic; Genes, Tumor Suppressor; Humans; Male; Mice; Models, Biological; Multiple Myeloma; PPAR gamma; Response Elements; Retinal Dehydrogenase; Spleen; Splenomegaly; Transcription Factors; Tretinoin; Vitamin A

Although CD138 expression is a hallmark of plasma cells and myeloma cells, reduced CD138 expression is occasionally found. However, the mechanisms underlying CD138 downregulation in myeloma cells remain unclear. Previous reports suggest that the bone marrow microenvironment may contribute to CD138 downregulation. Among various factors in the tumor microenvironment, hypoxia is associated with tumor progression, poor clinical outcomes, dedifferentiation and the formation of cancer stem cell niches in solid tumors. Since recent findings showed that progression of multiple myeloma (MM) delivers hypoxia within the bone marrow, we hypothesized that CD138 expression may be regulated by hypoxia. In the present study, we examined whether the expression of CD138 and transcription factors occurred in myeloma cells under hypoxic conditions. MM cell lines (KMS-12BM and RPMI 8226) were cultured under normoxic or hypoxic conditions for up to 30 days. Changes in the phenotype and the expression of surface antigens and transcription factors were analyzed using flow cytometry, RT-PCR and western blotting. All-trans retinoic acid (ATRA) was used to examine the phenotypic changes under hypoxic conditions. The expression levels of CD138, CS1 and plasma cell-specific transcription factors decreased under hypoxic conditions, while those of CD20, CXCR4 and B cell-specific transcription factors increased compared with those under normoxic conditions. Stem cell-specific transcription factors were upregulated under hypoxic conditions, while no difference was observed in ALDH activity. The reduced CD138 expression under hypoxic conditions recovered when cells were treated with ATRA, even under hypoxic conditions, along with decreases in the expression of stem cell-specific transcription factor. Interestingly, ATRA treatment sensitized MM cells to bortezomib under hypoxia. We propose that hypoxia induces immature and stem cell-like transcription phenotypes in myeloma cells. Taken together with our previous observation that decreased CD138 expression is correlated with disease progression, the present data suggest that a hypoxic microenvironment affects the phenotype of MM cells, which may correlate with disease progression.

Topics: Aldehyde Oxidoreductases; Antigens, CD20; Antineoplastic Agents; Apoptosis; Boronic Acids; Bortezomib; Cell Differentiation; Cell Hypoxia; Cell Line, Tumor; Down-Regulation; Humans; Intercellular Signaling Peptides and Proteins; Multiple Myeloma; Neoplastic Stem Cells; Oxygen; PAX5 Transcription Factor; Peptides; Pyrazines; Receptors, CXCR4; Syndecan-1; Tretinoin; Tumor Microenvironment

It is of clinical importance to find methods to overcome bortezomib resistance. In the current study, we clarified the relationship between resistance to bortezomib and the differentiation status of myeloma cells, and explored the feasibility of induction of differentiation in overcoming bortezomib resistance in myeloma.. Cell morphology, immunoglobulin light-chain protein secretion levels, and XBP-1 expression were used to evaluate the differentiation status of myeloma cells. Low dose 2-ME2 alone or in combination with ATRA was used to induce differentiation in myeloma cells.. The differentiation status of myeloma cells was related to myeloma sensitivity to bortezomib. After successful induction of differentiation, the myeloma cells were more sensitive to bortezomib with decreased growth and an increased rate of apoptosis. Induction of differentiation increased the proteasome workload in myeloma cells by increasing immunoglobulin secretion, while reducing proteasome capacity by decreasing proteasome activity. The imbalance between increased proteasome workload and decreased proteasome capacity is a possible mechanism by which induction of differentiation overcomes myeloma resistance to bortezomib.. The current study demonstrated, for the first time, that myeloma differentiation status is associated with myeloma sensitivity to bortezomib and that induction of differentiation can overcome myeloma resistance to bortezomib.

Topics: 2-Methoxyestradiol; Antineoplastic Agents; Boronic Acids; Bortezomib; Cell Differentiation; DNA-Binding Proteins; Drug Resistance, Neoplasm; Estradiol; Humans; Multiple Myeloma; Pyrazines; Regulatory Factor X Transcription Factors; Transcription Factors; Tretinoin; Tumor Cells, Cultured; X-Box Binding Protein 1

To observe the effect of rosiglitazone (RGZ) and all-trans-retinoic acid (ATRA) on the growth of myeloma xenograft in nude mice and to explore the influence of RGZ and ATRA on VEGF expression and angiogenesis in the tumor.. VEGF gene expression in myeloma cell line U266 cells was analyzed by semi-quantitative RT-PCR after incubation with RGZ, ATRA, or RGZ + ATRA for 24 h. Myeloma xenograft was established by subcutaneous injection of 10(7) U266 cells in the scapula area of 4-week old nude mice. 7 days later, the nude mice were administered with RGZ, ATRA or RGZ + ATRA, respectively, by intraperitoneal injection once every day for 21 days. The control mice were given equal volume of normal saline instead of the drug. On the 21(st) day of treatment, the mice were sacrificed and the tumors were taken off, and the tumor volume and weight were measured. The tumors were examined by histopathology with HE staining, and microvessel density (MVD), CD34 and VEGF expression in the tumors were analyzed by immunohistochemical staining.. VEGF mRNA was highly expressed in U266 cells and was decreased in a dose-dependent manner after incubation with RGZ. The VEGF mRNA level was further more decreased after RGZ + ATRA treatment. Xenografts of U266 cells were developed in all nude mice. The volume and weight of xenografts in the RGZ group were (785 ± 262) mm(3) and (1748 ± 365) mg, respectively, significantly lower than those of the control group (both P < 0.01). More significant inhibition was in the RGZ + ATRA group, (154 ± 89) mm(3) and (626 ± 102) mg, respectively, both were P < 0.05 vs. the RGZ group. RGZ inhibited the angiogenesis in U266 xenografts and immunohistochemical staining showed that the tumor MVD and VEGF expression were significantly decreased by RGZ treatment, and further more inhibited in the RGZ + ATRA group. VEGF protein was expressed in all xenografts in the nude mice. Its immunohistochemical staining intensity was 2.20 ± 0.40 in the control group, significantly higher than that of 1.48 ± 0.37 in the RGZ group (P < 0.01), and that of RGZ + ATRA group was 0.58 ± 0.26, further significantly lower than that of the RGZ group (P < 0.01). CD34 was expressed in all xenografts, most highly in the control group and lowest in the RGZ + ATRA group. The microvessel density (MVD) was highest in the control group (56.4 ± 15.2), significantly lower in the RGZ group (44.6 ± 11.2) (P < 0.05), and lowest in the RGZ + ATRA group (21.5 ± 8.6, P < 0.01).. The growth of myeloma cells can also be inhibited by RGZ and ATRA in nude mice in vivo. In addition to differentiation and apoptosis induction, RGZ can inhibit the formation of myeloma xenograft probably also through the downregulation of VEGF expression and subsequent angiogenesis.

Topics: Animals; Antigens, CD34; Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Cell Proliferation; Dose-Response Relationship, Drug; Drug Synergism; Female; Humans; Mice; Mice, Inbred BALB C; Mice, Nude; Microvessels; Multiple Myeloma; Neoplasm Transplantation; Neovascularization, Pathologic; RNA, Messenger; Rosiglitazone; Thiazolidinediones; Tretinoin; Tumor Burden; Vascular Endothelial Growth Factor A; Xenograft Model Antitumor Assays

Activation of PPARgamma by its ligands has shown differentiating effects in solid tumors. However, few reports addressed its role in myeloma cells. Our study demonstrated that exposure to PPARgamma ligand (rosiglitazone, RGZ) induced proliferation inhibition and cell cycle arrest in myeloma cells. A combination of RGZ with all-trans retinoic acid (ATRA) can enhance the growth inhibition effects of RGZ. Further study shows that RGZ-treated myeloma cells displayed morphological characteristics of cell differentiation, and more evident signs of differentiation were observed when RGZ was combined with ATRA. These changes were confirmed by the detection of CD49e expression and light chain protein secretion. Similar results were also observed when primary CD138(+) cells were treated with RGZ and ATRA. Collectively, our study revealed that RGZ can induce cell differentiation in myeloma cells and concomitant treatment with ATRA can enhanced the effects of RGZ.

Topics: Aged; Animals; Antineoplastic Agents; Cell Cycle; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Cell Shape; Dose-Response Relationship, Drug; Enzyme-Linked Immunosorbent Assay; Female; Flow Cytometry; Humans; Immunoglobulin Light Chains; Integrin alpha5; Male; Middle Aged; Multiple Myeloma; PPAR gamma; Rosiglitazone; Syndecan-1; Thiazolidinediones; Tretinoin; Tumor Cells, Cultured

Specific genetic alterations in multiple myeloma (MM) may cause more aggressive diseases. Paired gene array analysis on 51 samples showed that retinoic acid (RA) receptor alpha (RARalpha) expression significantly increased at relapse compared with diagnosis. RARalpha encodes 2 major isoforms: RARalpha1 and RARalpha2. In this study, we examined the function of RARalpha2 in MM. Reverse transcription-polymerase chain reaction (RT-PCR) revealed ubiquitous RARalpha1 expression in MM cells, but RARalpha2 was expressed in 26 (32%) of 80 newly diagnosed patients and 10 (28%) of 36 MM cell lines. Patients with RARalpha2 expression had a significantly shorter overall survival on identical treatments. The presence of RARalpha2 remained significant on multivariate analysis. Knockdown of RARalpha2 but not RARalpha1 induced significant MM cell death and growth inhibition, and overexpressing RARalpha2 activated STAT3 and mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling pathways. Interestingly, all-trans retinoic acid (ATRA) treatment induced potent cell death and growth inhibition in RARalpha2(+) but not RARalpha2(-) MM cells; overexpressing RARalpha2 in RARalpha2-deficient MM cells restored sensitivity to ATRA. Furthermore, ATRA treatment significantly inhibited the growth of RARalpha2-overexpressing MM tumors in severe combined immunodeficiency (SCID) mouse model. These findings provide a rationale for RA-based therapy in aggressive RARalpha2(+) MM.

Topics: Animals; Cell Proliferation; Disease Progression; Drug Resistance, Neoplasm; Gene Expression Profiling; Humans; Male; Mice; Mice, SCID; Multiple Myeloma; Predictive Value of Tests; Protein Isoforms; Receptors, Retinoic Acid; Recurrence; Retinoic Acid Receptor alpha; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; STAT3 Transcription Factor; Survival Rate; Tretinoin; Tumor Cells, Cultured

Activation of PPARgamma by its ligands has shown potential anti-neoplastic effects in solid tumors. In this study, we investigate the effects of rosiglitazone (RGZ) alone as well as in combination with all trans-retinoic acid (ATRA) on human myeloma cell lines and try to address its potential mechanism.. U266, RPMI-8226 and primary myeloma cells from patients were treated with different concentrations of RGZ in the presence or absence of ATRA and various biological responses were studied by the methods of [3H] thymidine incorporation, MTT, cell cycle analysis, Annexin V-PI staining, Wright-Giemsa staining, CD49e expression assay, light chain protein detection, RT-PCR and caspase-3 activity assay.. We report that exposure to RGZ induced proliferation inhibition and viability reduction in a dose-dependent manner in both U266 and RPMI-8226 cells. A similar exposure to RGZ also induced cell cycle arrest and cell apoptosis of myeloma cells. A combination of RGZ with ATRA enhanced the effects of RGZ and induced cell cycle arrest and apoptosis more profoundly in both cell lines. RGZ treated cells displayed morphological characteristics of cell differentiation, and more evident signs of differentiation were observed when combined with ATRA. These changes were confirmed by the detection of CD49e expression and light chain protein secretion. Similar cell apoptosis and differentiation were observed when primary CD138+ myeloma cells were treated with RGZ and ATRA. The mRNA expressions of FLIP, XIAP and survivin were detected in both cell lines and the levels decreased significantly after culture with RGZ. The addition of ATRA in culture medium made these changes more apparently. Caspase-3 activity was increased upon exposure to RGZ in both U266 and RPMI-8226 cells while combination of RGZ and ATRA brought out more effective activation of caspase-3. Similar apoptosis and cell differentiation induced by RGZ and ATRA can also be observed in primary CD138+ cells from myeloma patients.. Concomitant RXRalpha activation by ATRA enhanced the inhibitory effects of RGZ on myeloma cell proliferation, cell cycle, apoptosis and differentiation. Combination of RGZ and ATRA may be a useful therapy for human multiple myeloma.

Topics: Aged; Apoptosis; Caspase 3; Cell Differentiation; Cell Line, Tumor; Cell Survival; Female; Humans; Hypoglycemic Agents; Male; Middle Aged; Multiple Myeloma; PPAR gamma; Rosiglitazone; Thiazolidinediones; Tretinoin

Tamibarotene (TM411) is a synthetic retinoic acid receptor-alpha/-beta selective retinoid that is chemically more stable than all-trans retinoic acid. This study was designed to evaluate the activity of TM411 in multiple myeloma (MM) and the effects of TM411 combined with a glucocorticoid (GC). In vitro, five human myeloma cells were treated with TM411 alone, GC alone, or TM411 + GC. Cell survival was analyzed by the tetrazolium dye assay and the Hoechst 33342/propidium iodide double-staining method. The effect of TM411 + GC was assessed by the isobologram method. In vivo, the growth-inhibitory effects of the drugs on RPMI-8226 cell xenografts established in SCID mice were examined. The effects of the agents on IL-6-mediated signaling pathways were also analyzed by Western blotting. TM411 was 2- to 10-fold more potent, in terms of its growth-inhibitory effect, than all-trans retinoic acid. The combination of TM411 and GC was found to show a markedly synergistic interaction. While increased expressions of the IL-6 receptor, phosphorylated MAPK, and Akt were observed after exposure to GC, TM411 attenuated this increase in the expressions, suggesting that such modification of the effect of GC by TM411 might be the possible mechanism underlying the synergistic interaction. Furthermore, TM411 + GC showed a supra-additive inhibitory effect in a xenograft model as compared with TM411 or GC alone. These results imply that the combination of TM411 + GC might be highly effective against MM, and suggest the need for clinical evaluation of TM411 + GC for the treatment of MM.

Topics: Animals; Antineoplastic Agents; Benzoates; Cell Division; Cell Line, Tumor; Cell Survival; Drug Synergism; Female; Glucocorticoids; Humans; Mice; Mice, SCID; Multiple Myeloma; Retinoids; Tetrahydronaphthalenes; Tretinoin

To investigate the effects of artificial ligand of peroxisome proliferators activated receptors (PPARs), rosiglitazone (RGZ) and all trans-retinoic acid (ATRA) on human myeloma cell line growth in vitro and in vivo.. U266 and RPMI 8226 cells were treated with different concentration of RGZ in the presence or absence of ATRA and the results were studied by 3H-TdR thymidine incorporation (for cells proliferation), Annexin V-PI staining and caspase-3 activity assay (for cells apoptosis), RT-PCR (for FLIP, XIAP and survivin mRNA expression), and tumor formation test in BALB/c nude mice.. Exposure to RGZ induced proliferation inhibition in a dose-dependent manner in both U266 (r = 0.991, P < 0.01) and RPMI 8226 cells (r = 0.961, P < 0.01). A combination of RGZ with ATRA could enhance the inhibition effect (P < 0.001 in U266, P < 0.01 in RPMI8226). 10 micromol/L of RGZ induced apoptosis of (9.8 +/- 1.7)% in U266 cells and (10.7 +/- 3.3)% in RPMI8226 cells, in a time and dose dependent manner and combined with ATRA intensified the apoptosis induction effects (P < 0.01 in both cell lines). The FLIP, XIAP and survivin mRNAs were expressed in both cell lines and their levels decreased significantly after cultured with RGZ. The addition of RGZ + ATRA in the culture further decreased the levels. Caspase-3 activity increased substantially with the increase of RGZ concentration and the addition of RGZ + ATRA in the culture medium showed similar synergism effect on caspase-3 activation (P < 0.01). The xenograft of U266 cells in BALB/c nude mice were inhibited by RGZ and so did more by the combination of RGZ and ATRA (P < 0.01).. The down-regulation of FLIP, XIAP and Survivin induced by RGZ can activate caspase-3, whereby induced apoptosis and proliferation inhibition in myeloma cells. ATRA can enhance these effects of RGZ.

Topics: Animals; Antineoplastic Agents; Apoptosis; Caspase 3; Cell Line, Tumor; Cell Proliferation; Female; Humans; Inhibitor of Apoptosis Proteins; Mice; Mice, Inbred BALB C; Mice, Nude; Multiple Myeloma; Rosiglitazone; Signal Transduction; Thiazolidinediones; Tretinoin

To investigate the effects of PPARgamma ligand (rosiglitazone, RGZ) as well as combined with all trans-retinoic acid (ATRA) on human myeloma cells and try to explore the possible mechanism.. Human myeloma cell lines U266 and RPMI-8226 cells were treated with RGZ in the presence or absence of ATRA. Cell proliferation was evaluated by [(3)H] thymidine incorporation, cell cycle distribution and CD49e expression were analyzed by flow cytometry, morphology changes were evaluated by Wright-Giemsa staining, and p27(Kip1) and p21(Waf1) expression was detected by Western blotting.. The exposure to RGZ induced proliferation inhibition in both cell lines in a dose-dependent manner. After cultured with 5 micromol/L RGZ, the proportion of U266 and RPMI-8226 cells in phase G(0)/G(1) was (45.2 +/- 6.7)% and (40.3 +/- 7.3)%, respectively (P < 0.05). The proportion of the cells in phase G(2)/M and S was (52.2 +/- 7.4)% and (57.4 +/- 9.5)%, respectively (P < 0.05). These changes were more evident when the RGZ concentration was increased to 10 micromol/L. A combination of RGZ with ATRA enhanced the growth inhibition and cell cycle arrest effects of RGZ. The RGZ-treated myeloma cells displayed morphological characteristics of cell differentiation, and more evident signs of differentiation were observed when RGZ was combined with ATRA. These changes were confirmed by the detection of CD49e expression. The expression of p27(Kip1) and p21(Waf1) in myeloma cells was up-regulated by RGZ and this change was more apparent when RGZ was used in combination with ATRA.. RGZ can induce cell cycle arrest and cell differentiation in myeloma cells which maybe caused by up-regulation of p27(Kip1) and p21(Waf1) expression. ATRA can enhance these effects of RGZ on multiple myeloma cells and combined use of these two drugs may show a synergistic effect on myeloma cells.

Topics: Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Dose-Response Relationship, Drug; Drug Synergism; Humans; Integrin alpha5; Intracellular Signaling Peptides and Proteins; Multiple Myeloma; PPAR gamma; Rosiglitazone; Thiazolidinediones; Tretinoin; Up-Regulation

The nuclear arrangement of promyelocytic leukaemia nuclear bodies (PML NBs) was studied in vitro after the cell treatment by clinically used agents such as all-trans retinoic acid (RA) in human leukaemia and cytostatics or gamma radiation in multiple myeloma cells. In addition, the influence of phorbol ester (PMA) on PML NBs formation was analyzed. A reduced number of PML bodies, which led to relocation of PML NBs closer to the nuclear interior, mostly accompanied RA- and PMA-induced differentiation. Centrally located PML NBs were associated with transcriptional protein RNAP II and SC35 regions, which support importance of PML NBs in RNA processing that mostly proceeds within the nuclear interior. Conversely, the quantity of PML NBs was increased after cytostatic treatment, which caused re-distribution of PML NBs closer to the nuclear envelope. Here we showed correlations between the number of PML NBs and average Centre-to-PML distances. Moreover, a number of cells in S phase, especially during differentiation, influenced number of PML NBs. Studying the proteins involved in PML compartment, such as c-MYC, cell-type specific association of c-MYC and the PML NBs was observed in selected leukaemic cells undergoing differentiation, which was accompanied by c-MYC down-regulation.

Topics: Cell Cycle; Cell Differentiation; Cell Line, Tumor; Cell Nucleus; Flow Cytometry; Gamma Rays; HL-60 Cells; Humans; Intranuclear Inclusion Bodies; K562 Cells; Leukemia, Promyelocytic, Acute; Melphalan; Multiple Myeloma; Tetradecanoylphorbol Acetate; Tretinoin; U937 Cells

Hematological malignancies such as leukemia or lymphoma are mainly treated by hospitalization or in outpatient clinics. Therefore, home care and home nursing are not so intensively done in the treatment of these malignancies. However, G-CSF administration against neutropenia after chemotherapy and administration of narcotics or opioids against severe pain have been performed sometimes during home care, and have been contributing to better QOL of the patients.

Topics: Analgesics, Opioid; Granulocyte Colony-Stimulating Factor; Home Care Services, Hospital-Based; Humans; Leukemia; Lymphoma; Multiple Myeloma; Myelodysplastic Syndromes; Neutropenia; Pain; Quality of Life; Tretinoin

To look for new candidates for agents to use in maintenance therapy for myeloma patients, the growth inhibitory effects of a 3-hydroxy-3-mehtylglutaryl coenzyme A (HMG-CoA) reductase inhibitor (statin), simvastatin, was analyzed using human myeloma cell lines. Several investigations have indicated growth reduction in certain lineages of cancer cells including one report on myeloma, and inhibitory effects of statins on GTPases and involving MAP-kinases. Most (12 out of 13) myeloma lines examined showed growth inhibition when cultured with various concentrations (1-30 microM) of simvastatin in a dose-dependent manner. Simvastatin in combination with other biological response modifiers such as ATRA or DEX had additional effects on growth. In addition, anti-oxides prevented the simvastatin-induced growth inhibition and apoptosis. Furthermore, myeloma cells treated with simvastatin clearly showed inactivation of various MAP-kinase pathways such as ERK1/2, MEK1/2, JNK, and p38. Based on these findings, statins may be suitable for clinical usage in maintenance therapy for myeloma patients.

Topics: Antioxidants; Apoptosis; Cell Division; Cell Line, Tumor; Dexamethasone; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Interferon-alpha; JNK Mitogen-Activated Protein Kinases; MAP Kinase Kinase 1; MAP Kinase Kinase 2; MAP Kinase Kinase 4; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinase Kinases; Multiple Myeloma; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Simvastatin; Tretinoin

Since multiple myeloma responds poorly to conventional chemotherapy or radiotherapy, new therapeutic approaches are needed. This study investigated the effects of dexamethasone, all-trans retinoic acid (ATRA), the active metabolite of vitamin D(3) [1,25(OH)(2)D(3)] and interferon-alpha on FO mouse myeloma cells (non-immunoglobulin-secreting myeloma cell line) in single drug or drug combination groups in vitro.. Apoptosis ratio and change in cell counts in 4 single drug groups (dexamethasone, ATRA, vitamin D(3) and interferon-alpha) and 6 combination drug groups (dexamethasone + vitamin D(3,) dexamethasone + ATRA, dexamethasone + interferon-alpha, vitamin D(3) + ATRA, vitamin D(3) + interferon-alpha, interferon-alpha + ATRA) were compared with the control group.. When treatment groups were compared with the control group, there was a significant increase in apoptosis in all, but this was most prominent in the group treated with dexamethasone alone. The apoptosis ratios were 0.10 and 6.82% in the control and dexamethasone-only groups, respectively. We also found that there was a significant decrease in cell count, particularly in the dexamethasone-only, ATRA-only, and ATRA-vitamin D(3) combination groups.. ATRA, interferon-alpha, vitaminD(3) and particularly dexamethasone have significant effects on FO mouse myeloma cells resulting in a decreased cell count and an increased apoptosis ratio. This study should be repeated with human myeloma cell lines for further information.

Topics: Animals; Antineoplastic Agents; Antineoplastic Agents, Hormonal; Apoptosis; Cell Count; Cholecalciferol; Dexamethasone; Drug Interactions; Interferon-alpha; Mice; Multiple Myeloma; Tretinoin; Tumor Cells, Cultured

Multiple myeloma is essentially an incurable malignancy and it is therefore of great interest to develop new therapeutic approaches. We previously reported that human B cell-lymphomas express the nuclear receptor peroxisome proliferator-activated receptor gamma (PPARgamma) and are killed by PPARgamma ligands. Herein, we investigate the therapeutic potential of PPARgamma ligands for multiple myeloma. The human multiple myeloma cell lines ANBL6 and 8226 express PPARgamma mRNA and protein. The PPARgamma ligands, 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2) and ciglitazone, induced multiple myeloma cell apoptosis as determined by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay, loss of mitochondrial membrane potential, and caspase activation. Importantly, the ability of PPARgamma ligands to kill both multiple myeloma cell lines was not abrogated by Interleukin-6 (IL-6), a multiple myeloma growth survival factor. Finally, the RXR ligand 9-cis retinoic acid (9-cis RA) in combination with PPARgamma ligands greatly enhanced multiple myeloma cell killing. These new findings support that PPARgamma ligands may represent a novel therapy for multiple myeloma.

Topics: Apoptosis; Blotting, Western; Caspases; Cell Line, Tumor; Enzyme Activation; Humans; Immunohistochemistry; Immunologic Factors; In Situ Nick-End Labeling; Interleukin-6; Ligands; Membrane Potentials; Mitochondria; Multiple Myeloma; PPAR gamma; Prostaglandin D2; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Thiazolidinediones; Tretinoin

The synthetic retinoid 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (AHPN/CD437) appears to possess an apoptotic activity superior to classical retinoids in vitro as in vivo. Numerous studies have shown that CD437-induced apoptosis is independent of its nuclear receptor activity, suggesting that CD437 might have a unique mechanism of action. The purpose of this study was to compare CD437- and all-trans retinoic acid (atRA)-induced cell death. CD437 provoked a rapid apoptotic phenotype immediately followed by secondary necrosis in RPMI 8226, U266 and L363 human myeloma cell lines. Nuclear apoptotic features were observed upon both CD437 and atRA treatments. In contrast, membrane blebbing and the subsequent formation of apoptotic bodies, a classical apoptotic event, was only observed upon atRA treatment. In addition, CD437, contrary to atRA, was unable to induce tissue transglutaminase (tTG), an intracellular enzyme involved in the formation of cross-linked protein polymers contributing to apoptotic morphological changes. Taken together, these data suggest that CD437 induces rapid but incomplete apoptotic phenotype in human myeloma cells.

Topics: Antineoplastic Agents; Apoptosis; Cell Survival; Fluorescent Antibody Technique, Indirect; Humans; Lamin Type B; Multiple Myeloma; Retinoids; RNA, Messenger; Transglutaminases; Tretinoin; Tumor Cells, Cultured; Ultrasonography

Recently, it was disclosed that all-trans retinoic acid (ATRA) inhibits myeloma cell growth by downregulating the interleukin 6 (IL-6)/IL-6 receptor (IL-6R) auto/paracrine loop, and upregulating p21/Cip1 cyclin-dependent kinase inhibitor (CDK-I), thereby inducing apoptosis with a decrease in Bcl-2 protein expression. To elucidate and generalize the effects of ATRA on the proliferation and cellular biology of myeloma cells, 12 human myeloma cell lines established in our laboratory were utilized. Two out of the 12 lines showed enhanced growth on supplementation of ATRA and were characterized by IL-10 production, downregulation of membrane Fas and reduced upregulation of p21/Cip1 CDK-I message. These characteristics may prove important for the clinical use of ATRA and should be considered before starting ATRA therapy for myeloma.

Topics: Antibodies, Monoclonal; Apoptosis; CDC2 Protein Kinase; Cell Adhesion Molecules; Cell Division; Dose-Response Relationship, Drug; fas Receptor; Flow Cytometry; Humans; Interleukin-10; Multiple Myeloma; Reverse Transcriptase Polymerase Chain Reaction; Tretinoin; Tumor Cells, Cultured

Bisphosphonates (BPs) are effective in the management of bone disease in patients with multiple myeloma. Recent reports have suggested that they may also have an antitumor activity. YM529 is a new synthetic BP with more than 1000 times the bone resorption inhibitory activity of pamidronate. To clarify the direct effects of YM529 on myeloma cells, the cell proliferation and cell cycle perturbation were analyzed using 12 myeloma cell lines established in our laboratory. The growth inhibition was dose dependent. The cells accumulated in [2n<<4n] of the cell cycle and subsequently formed an apoptotic sub-G1 fraction. Combined treatment with all-trans retinoic acid, thalidomide, or interferon-alpha enhanced the growth inhibitory effects of YM529 on these cells. However, there were no remarkable effects of YM529 on the messenger RNA expression for angiogenic factors, cell cycle regulators, or cytokines related to myeloma cells. These results indicate that YM529 is beneficial not only to bone lesions but also for its direct antitumor effects on myeloma cells.

Topics: Antineoplastic Combined Chemotherapy Protocols; Cell Division; Diphosphonates; Drug Synergism; Humans; Imidazoles; Interferon-alpha; Interphase; Multiple Myeloma; RNA; Thalidomide; Tretinoin; Tumor Cells, Cultured

The 5T33 murine model of multiple myeloma was used to investigate the potential of all-trans retinoic acid (ATRA) to purge clonogenic myeloma cells from autologous hemopoietic stem-cell harvests by differentiating immature 5T33 cells into terminal-stage plasma cells with limited repopulation capacity.. 5T33 cells were treated with 10 microM ATRA and the effect on cell clonogenicity was determined by measuring the time to paraprotein detection in C57Bl/KaLwRij mice compared to control animals. Cell differentiation and apoptosis following ATRA treatment were investigated using flow cytometry and caspase-3 assay. Treatment with ATRA resulted in a 33% reduction in the in vitro cloning efficiency of 5T33 cells. Reduced in vitro clonogenicity of 5T33 cells following ATRA treatment was supported by a 16-49% increase in the time taken for C57Bl/KaLwRij mice to develop paraprotein following injection of 5T33 cells pretreated with ATRA for 8 days. Although ATRA was shown not to alter the in vitro growth characteristics of 5T33 cells, significant inhibition of apoptosis was observed.. Treatment with ATRA also resulted in an increase in the proportion of 5T33 cells expressing the CD54 adhesion molecule, which is known to be highly expressed on mature myeloma cells.. The ability of ATRA to decrease the clonogenicity of 5T33 cells in vitro and increase the time to disease development in vivo suggests that this drug may be useful for purging autologous stem cell harvests in the clinical setting.

Topics: Animals; Apoptosis; Bone Marrow Purging; Cell Differentiation; Cell Division; Hematopoietic Stem Cell Transplantation; Hematopoietic Stem Cells; Intercellular Adhesion Molecule-1; Mice; Mice, Inbred C57BL; Models, Animal; Multiple Myeloma; Myeloma Proteins; Neoplasm Transplantation; Neoplastic Stem Cells; Plasma Cells; Transplantation, Autologous; Tretinoin; Tumor Cells, Cultured; Tumor Stem Cell Assay

The bone marrow micro-environment produces a number of different survival factors that are important for the malignant growth and drug resistance of multiple myeloma (MM) cells. One of the main factors reported to be essential for survival and growth of MM cells in some experimental systems is IL-6. Therefore, the development and testing of substances that interfere with IL-6 or IL-6 receptor (IL-6R) function might have therapeutic value for the treatment of MM. We analyzed the effect of the IL-6R antagonist SANT-7 on growth and survival of the IL-6--dependent MM cell lines INA-6 and XG-1 as well as primary MM cells from 7 patients co-cultured with bone marrow stromal cells (BMSCs). In particular, we were interested in whether SANT-7 enhances the growth-inhibitory effects of dexamethasone (Dex) and all-trans-retinoic acid (ATRA). None of the drugs when tested as a single substance, including SANT-7, induced major growth inhibition if MM cells were co-cultured with primary human BMSCs. However, when Dex and ATRA were given in combination with SANT-7, strong growth inhibition was achieved in cell lines and primary MM cells. This effect was due to cell-cycle arrest and induction of apoptosis.

Topics: Antineoplastic Agents; Apoptosis; Bone Marrow Cells; Cell Cycle; Cell Division; Dexamethasone; Drug Resistance, Neoplasm; Drug Screening Assays, Antitumor; Fluorescent Antibody Technique; Humans; Interleukin-6; Multiple Myeloma; Receptors, Interleukin-6; Stromal Cells; Tretinoin; Tumor Cells, Cultured

The positions of nucleosomes in the proximal 5' regions of the coordinately regulated murine entactin/nidogen and laminin gamma1 genes have been identified in four different transcriptional states--constitutively off, basal, induced, and constitutively induced. In the entactin gene a 450 base pair (bp) region of open chromatin is present between three positioned nucleosomes and the transcriptional start site in the basal, induced, and constitutively induced states. Additionally there is a 200 bp open chromatin region at approximately -2.1 kbp that is only present in the induced and constitutively induced states. In the laminin gamma1 gene, a 650 bp region of nucleosome-free chromatin is present between nucleosomes positioned at approximately -750 and +120 in all transcriptionally active states. These results suggest that basal co-expression of these genes requires sites present in these near upstream regions. The induction to high levels appears to involve additional sites and possibly the production of new and/or the modification of existing trans-acting factors.

Topics: Animals; Bucladesine; Cell Differentiation; Chromatin; Chromosome Mapping; Gene Expression Regulation, Neoplastic; Genes; Laminin; Membrane Glycoproteins; Mice; Multiple Myeloma; Neoplasm Proteins; Nucleosomes; Teratocarcinoma; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

To investigate the response of multiple myeloma (MM) cells to arsenic trioxide (As2O3) and their possible mechanisms.. Two MM-derived cell lines RPMI8226 and U266 cells were used as in vitro models. Cell apoptosis was assessed by morphology, flow cytometry, and DNA gel electrophoresis. Mitochondrial transmembrane potentials (delta psi m) were evaluated by measuring cellular Rhodamine 123 staining intensity. Protein expression was analyzed using Western blot.. Zero point one to 0.5 mumol/L As2O3 inhibited cell proliferation and 2.0 mumol/L As2O3 induced cell apoptosis, while 1.0 mumol/L As2O3 inhibited proliferation with a weak degree of apoptosis induction in RPMI8226 and U266 cell lines. As2O3-induced apoptosis was accompanied by mitochondrial transmembrane potentials (delta psi m) collapse and caspase-3 activation in the presence of intact membrane. Glutathione depleter buthionine sulfoximine enhanced, while disulfide bond-reducing agent dithiothreitol partially antagonized As2O3-induced delta psi m collapse and apoptosis in MM cells. All-trans retinoic acid (ATRA) could also induce apoptosis in RPMI8226 cells, but it did not show any cooperative effects with As2O3.. As2O3 exerts apoptosis-inducing and growth-inhibiting effects on MM cells, and mitochondrium is a pivotal and common target of As2O3 for apoptosis induction.

Topics: Antineoplastic Agents; Apoptosis; Arsenic Trioxide; Arsenicals; Buthionine Sulfoximine; Caspase 3; Caspases; Dose-Response Relationship, Drug; Enzyme Activation; Humans; Membrane Potentials; Mitochondria; Multiple Myeloma; Oxides; Tretinoin; Tumor Cells, Cultured

To investigate the possible mechanisms of arsenic trioxide (As2O3)-induced apoptosis in multiple myeloma (MM) cell line, and the interactions between As2O3 and all-trans retinoic acid (ATRA) or interferon(IFN)-alpha.. Multiple myeloma cell lines RPMI 8226 and U266 were treated with As2O3 in combination with dithiothreitol (DTT), buthionine sulfoximine (BSO), ATRA and IFN-alpha. Cell viability was counted by trypan-blue exclusion. Apoptosis was assessed by cell morphology and flow cytometry. Mitochondrial transmembrane potentials (delta psi m) were measured by cellular rhodamine 123 staining intensity on flow cytometry.. Glutathione depleting agent BSO at 1.0 mmol/L enhanced, while disulfide bond-reducing agent DTT at 0.2 mmol/L partially antagonized As2O3-induced decline of delta psi m and apoptosis in MM cells. ATRA also induced RPMI 8226 cell apoptosis, but it could not synergize with As2O3. On the other hand, As2O3-induced apoptosis did not occur in U266 cells. In addition, IFN-alpha itself did not inhibit the growth and viability of MM cells, nor did it influence the effects of As2O3 on MM cells.. As2O3-induced apoptosis of RPMI 8226 multiple myeloma cells involves sulphyhydryl groups. ATRA and IFN-alpha do not synergize with As2O3 in the induction of apoptosis of MM cells.

Topics: Antineoplastic Agents; Apoptosis; Arsenic Trioxide; Arsenicals; Buthionine Sulfoximine; Cell Division; Dithiothreitol; Drug Interactions; Humans; Interferon-alpha; Multiple Myeloma; Oxides; Tretinoin; Tumor Cells, Cultured

All-trans retinoic acid (ATRA) has been shown to inhibit in vitro growth of multiple myeloma (MM) cells, and this effect can be further potentiated by the addition of Dexamethasone (DEX). We here extended this study by testing the activity of 9-cis retinoic acid (9-cis RA) and 13-cis retinoic acid (13-cis RA), both alone and in combination with DEX, in two MM cell lines, U266 and RPMI 8226. Furthermore, we aimed at investigating the mechanisms involved in the interactions of retinoids and DEX in this setting. 9-cis RA appeared to be the most active agent in U266 cell line (IC50 = 1.2 mumol/l vs 10.5 and 9.8 mumol/l obtained with ATRA and 13-cis RA, respectively) while, in RPMI 8226 cell line, 9-cis RA and 13-cis RA were almost equally cytotoxic (IC50 = 1 and 0.8 mumol/l) and ATRA was less effective. Co-incubation with DEX resulted in a synergistic cytotoxic activity in both the cell lines except for the combinations DEX + 9-cis RA in U266 cell line and DEX + 13-cis RA in RPMI 8226 cell line, where the effect was merely additive. A synergistic cytotoxic effect of retinoids and DEX was also observed on fresh MM cells obtained from 7 patients. Both retinoids and DEX are known to be inducers of apoptosis; we verified that the combined inhibitory activity of retinoids and DEX could be attributed to an increased induction of apoptosis. This effect may be mediated by a reduced intracellular expression of BCL-2 protein, which indeed observed after prolonged in vitro treatment with retinoids. It has been described recently that an enhanced expression of BCL-2 protein can contribute to the occurrence of early chemoresistance; the downregulation of BCL-2 protein induced by retinoids could thus be exploited, by means of novel chemotherapy plus retinoids combinations, in order to improve the efficacy of conventional chemotherapy in MM.

Topics: Alitretinoin; Apoptosis; Dexamethasone; Drug Synergism; Genes, bcl-2; Humans; Isotretinoin; Multiple Myeloma; Proto-Oncogene Proteins c-bcl-2; Tretinoin; Tumor Cells, Cultured

All-trans retinoic acid (ATRA) has previously been shown to inhibit the growth of OPM-2 human myeloma cells. The growth inhibition was postulated to result from a transcriptional downregulation of interleukin-6 receptor alpha (IL-6Ralpha) with IL-6Rbeta (gp130) unaffected. To formally test this hypothesis, an expression vector designed for constitutive IL-6Ralpha expression was constructed and used for transfection of OPM-2 cells. Six stable transfectants were cloned. The expression of IL-6Ralpha was shown by immunofluorescence with anti-IL-6Ralpha antibody and 125I-IL-6 binding. In five of six transfectant clones, cellular IL-6Ralpha was 1.5- to 6-fold higher than the parental cells, with the ligand binding affinity unchanged. While ATRA reduced IL-6Ralpha expression in the parental OPM-2 cells, it enhanced its expression in these five transfectants. The clonogenic growth of these transfectants, however, remained strongly inhibited by ATRA. Further analysis, comparing the parental OPM-2 cells and a representative transfectant, clone C5, showed that IL-6 caused rapid tyrosine phosphorylation of gp130 in both OPM-2 and C5 clones. Pretreatment with ATRA greatly reduced IL-6-induced gp130 phosphorylation in OPM-2 cells, reflecting a reduction in cellular IL-6Ralpha. In contrast, IL-6-induced gp130 phosphorylation was not reduced by ATRA pretreatment in C5 cells, indicating that the expressed IL-6Ralpha was functional. Similar to OPM-2 cells, C5 cells were sensitive to growth inhibition by dexamethasone, which was entirely reversed by exogenous IL-6, suggesting that the IL-6 postreceptor signal transduction remained intact. ATRA was further shown to upregulate p21(WAF1) expression and cause dephosphorylation of the retinoblastoma protein (pRB) in both OPM-2 and C5 cells. Exogenous IL-6 also failed to reverse these effects of ATRA. Thus, the growth inhibitory activity of ATRA is not mediated through cellular IL-6Ralpha downregulation and is likely to result from a direct upregulation of p21(WAF1) and consequent dephosphorylation of pRB.

Topics: Antineoplastic Agents; Cell Division; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Down-Regulation; Humans; Multiple Myeloma; Receptors, Interleukin-6; Signal Transduction; Transfection; Tretinoin; Tumor Cells, Cultured; Up-Regulation

We investigated the effects of all-trans retinoic acid (RA) and dexamethasone (Dex) on the in vitro growth of the human myeloma cell line RPMI 8226. RA inhibited RPMI 8226 cell growth by both antiproliferative effect and induction of apoptosis. Typical morphological and biochemical characteristics of apoptosis including chromatin condensation, apoptotic bodies formation and internucleosomal DNA cleavage were detected after 4 days of treatment with 1 microM RA. In situ TUNEL assay demonstrated that DNA cleavage preceded chromatin condensation. The expression of tissue transglutaminase (tTG), an enzyme proposed to play a role in apoptosis was induced with RA, as shown by both enzymatic assay and in situ immunofluorescence detection. Dex, when used alone, had no effect on cell growth and apoptosis. When combined to RA, Dex did not interfere with the RA-dependent inhibition of cell proliferation, but unexpectedly inhibited both quantitatively and qualitatively several morphological and biochemical features of the apoptosis induced by RA. Dex did not affect RA-induced DNA breaks formation but impeded the progression of chromatin condensation and the formation of apoptotic bodies. Interestingly, Dex also inhibited the RA-dependent induction of tTG. RU486, a glucocorticoid antagonist, counteracted all Dex effects. Taken together these data demonstrate that key cytoplasmic and nuclear events occurring during apoptosis are differentially regulated by RA and Dex in myeloma cell line RPMI 8226.

Topics: Apoptosis; Cell Division; Dexamethasone; DNA Fragmentation; Flow Cytometry; GTP Phosphohydrolases; GTP-Binding Proteins; Humans; Multiple Myeloma; Protein Glutamine gamma Glutamyltransferase 2; Transglutaminases; Tretinoin; Tumor Cells, Cultured

The association of leukemia and multiple myeloma is well described usually as a complication of chemotherapy but also in the absence of chemotherapy or at diagnosis. Such leukemias are typically acute myeloid leukemia (AML), particularly myelomonocytic subtype, and cases of acute promyelocytic leuke (APL) are rarely reported. Controversy exists as to whether myeloma and AML originate from a single haematopoietic progenitor or arise from different cell lineages. We report a case of a 58 year old female who developed APL 10 months following diagnosis of nonsecretory light chain (kappa) myeloma which had been treated with local spinal irradiation and low dose oral melphalan and prednisone. Clonality had originally been demonstrated by light chain restriction (kappa) of her bone marrow plasma cells whilst immunoglobulin heavy chain and T cell receptor genes were germ line. At development of APL cytogenetics revealed t(15;17) and PML-RAR fusion gene was detected by RT-PCR. The patient was treated with all-trans retinoic acid (ATRA) and received 2 cycles of consolidation chemotherapy with Idarubicin. Following this therapy the t(15;17) and PML-RAR were both undetectable whilst the clonal population of kappa staining plasma cells persisted. This particular patient represents a rare case of APL complicating multiple myeloma with persistence of the myeloma clone but disappearance of PML-RAR alpha RNA following therapy. This case study appears to support the argument that the APL and myeloma originated from distinct cell lineages.

Topics: Antibiotics, Antineoplastic; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Biomarkers, Tumor; Cell Lineage; Chromosomes, Human, Pair 15; Chromosomes, Human, Pair 17; Clone Cells; Combined Modality Therapy; Diphosphonates; Embryonal Carcinoma Stem Cells; Female; Gene Rearrangement, B-Lymphocyte, Light Chain; Humans; Idarubicin; Immunoglobulin kappa-Chains; Leukemia, Promyelocytic, Acute; Melphalan; Middle Aged; Multiple Myeloma; Myeloma Proteins; Neoplasm Proteins; Neoplasms, Multiple Primary; Neoplastic Stem Cells; Oncogene Proteins, Fusion; Osteolysis; Pamidronate; Prednisone; Remission Induction; Translocation, Genetic; Tretinoin

Retinoic acid and dexamethasone, in combination, inhibit the growth of human myeloma cell lines in a synergistic manner. Previously, we observed that all-trans retinoic acid (ATRA) caused G1 arrest and inhibited clonogenic growth of the OPM-2 human myeloma cell line. This was associated with downregulation of the IL-6 receptor (IL-6R) gp80 protein, while autocrine IL-6 production and gp130 were not affected. Growth inhibition was not reversed by the addition of exogenous IL-6 or forced, constitutive expression of the IL-6 receptor gp80 protein, suggesting that the mechanism of action of ATRA may be due to effects on the post-receptor pathway. Therefore, in this study we have investigated whether growth arrest was associated with changes in the level of phosphorylation of the RB protein. ATRA decreased the level of phosphorylation of the RB protein at doses > 5 x 10(-9) M and also induced a five fold increase in p21WAF1, while levels of p27KIP1 and CDK2 were unchanged. The ATRA-mediated increase in p21 preceded the change in RB phosphorylation and G1 arrest and was not reversed by the addition of exogenous IL-6. The levels of CDK2 activity were inhibited approximately 60% in ATRA-treated cells, suggesting that the increased p21 levels were sufficient to inhibit CDK activity and cause RB hypophosphorylation. Increased levels of p21 have recently been observed in human myeloma cells exposed to dexamethasone, and we suggest that the common ability of these two agents to inhibit myeloma cell growth depends on their induction of p21.

Topics: Antineoplastic Agents; Cell Division; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Humans; Multiple Myeloma; Phosphorylation; Retinoblastoma Protein; Signal Transduction; Tretinoin; Tumor Cells, Cultured; Up-Regulation

All-trans retinoic acid (ATRA) is a vitamin A derivative that induces the differentiation of myeloid leukemia cells in vitro and in vivo. Several investigators have recently reported that ATRA downregulates the production of interleukin-6 (IL-6) and the expression of IL-6 receptor (IL-6R) and also inhibits the proliferation of myeloma cells. It has also been reported that myeloma cells express Fas antigen, and in some of these cells apoptosis was induced by treatment with anti-Fas monoclonal antibody (mAb). In the present study, we demonstrated that ATRA increased Fas expression in the human myeloma cell line, U266B1. We observed that both apoptosis induction and growth inhibition were enhanced in cells exposed to a combination of anti-Fas mAb and ATRA compared with cells exposed to either treatment alone. We also examined whether ATRA modulated bcl-2, an anti-apoptosis protein, in U266B1 cells. Flow cytometry analysis revealed that the mean fluorescence intensity of bcl-2 protein was slightly decreased in cells treated with ATRA. These results indicate that in U266B1 cells, combined treatment with anti-Fas mAb and ATRA enhances the induction of apoptosis by modulating the expression of Fas and bcl-2 by ATRA.

Topics: Antibodies, Monoclonal; Apoptosis; Cell Division; fas Receptor; Humans; Keratolytic Agents; Multiple Myeloma; Tretinoin; Tumor Cells, Cultured

The effects of all trans retinoic acid (ATRA) on human myeloma cells growth were studied in vitro and in vivo using immunodeficient mice engrafted with malignant plasma cells. ATRA inhibited the in vitro proliferation of plasma cells originating from two patients with multiple myeloma whereas it had no effect on the in vivo growth of plasma cell grafts as assessed by the serial study of human Ig levels in mouse serum. Thus, the efficacy of ATRA for the treatment of human multiple myeloma remains to be ascertained.

Topics: Animals; Cell Division; Humans; Immunoglobulin G; Mice; Mice, SCID; Multiple Myeloma; Neoplasm Transplantation; Tretinoin; Tumor Cells, Cultured

Interleukin 6 (IL-6) is the most important known growth factor for multiple myeloma, and IL-6 signalling pathways are potential targets for therapy. We hypothesized that interfering with the IL-6 signalling pathway at more than one level would be more effective than a single block in inhibiting proliferation of myeloma cells. Accumulating data support the concept that glucocorticoids down-regulate IL-6, whereas retinoic acid derivatives (RA) down-regulate IL-6R in myeloma. We found that all-trans RA (ATRA), 13-cis-RA and 9-cis-RA each similarly inhibited growth of RPMI 8226 myeloma cells and that addition of dexamethasone (DEX) added to RA growth inhibition. The major effects of retinoids were to reduce the proliferative fraction and induce apoptosis whereas DEX increased the apoptotic fraction. When combined, apoptosis was enhanced. Effects of RA + DEX were also least able to be overcome by exogenous IL-6. RA decreased IL-6R levels and addition of DEX to RA delayed recovery of IL-6R levels compared with RA alone. Since RPMI 8226 cells have undetectable IL-6, we investigated U266B1 cells and found that RA and DEX decreased both IL-6 secretion and IL-6 RNA levels. Mechanistically, IL-6R down-regulation by RA was enhanced by DEX, whereas IL-6 protein and RNA levels were reduced by DEX and by RA. In summary, combinations of RA + DEX were not only more effective in inhibiting myeloma cells growth by the dual mechanisms of decreasing proliferative fraction and increasing apoptotic fraction, but were also less able to be overcome by IL-6.

Topics: Antineoplastic Agents; Antineoplastic Agents, Hormonal; Apoptosis; Cell Division; Dexamethasone; Dose-Response Relationship, Drug; Down-Regulation; Drug Synergism; Glucocorticoids; Humans; Interleukin-6; Multiple Myeloma; Receptors, Interleukin-6; Tretinoin; Tumor Cells, Cultured

Interleukin-6 (IL-6)/IL-6 receptor (IL-6R) plays a major role in autocrine/paracrine growth regulation of myeloma cells. We investigated the effect of dexamethasone and all-trans retinoic acid, previously shown to modulate IL-6/IL-6R, on the in vitro growth of a human myeloma cell line, OPM-2. Both agents inhibited the clonogenic growth and 3H-thymidine incorporation in a concentration-dependent fashion. Isobologram and median effect analysis showed a strong synergy between these two agents with a combination index in the range of 0.2 to 0.6. Both agents decreased the labeling index and the cell fraction in S and G2/M phases, suggesting a block in G1-S phase transition. The clonogenic growth was stimulated by exogenous IL-6 and was inhibited by monoclonal antibody to IL-6, suggesting an autocrine function of IL-6. The effect of dexamethasone but not all-trans retinoic acid was completely reversed by exogenous IL-6. Dexamethasone increased, while all-trans retinoic acid reduced, IL-6R but not gp130 mRNA expression. Their combination caused a net reduction in IL-6R mRNA. Cellular IL-6R density was altered correspondingly without changes in the binding affinity. IL-6 mRNA expression was reduced by dexamethasone and the combination, but was not affected by retinoic acid alone. However, IL-6 secretion into culture supernatant was abolished by both agents. A survey of 4 additional human myeloma cells showed that 1 was sensitive to both, 1 was sensitive to one agent only, and 2 were resistant to both. The study demonstrates the possibility of regulating myeloma cell growth through modulation of IL-6/IL-6R autocrine/paracrine loop and the principle of achieving a synergistic effect by blocking this loop at multiple sites.

Topics: Antineoplastic Agents; Base Sequence; Cell Cycle; Cell Division; Dexamethasone; Drug Synergism; Growth Inhibitors; Humans; Interleukin-6; Mitotic Index; Molecular Sequence Data; Multiple Myeloma; Recombinant Proteins; Tretinoin; Tumor Cells, Cultured; Tumor Stem Cell Assay

We previously showed that IL-6 is an autocrine growth factor for two human myeloma cell lines, RPMI 8226 and U266. We investigated here the in vitro and in vivo effects of all-trans retinoic acid (RA) on the growth and survival of these two cell lines. RA induced a dramatic dose- and time-dependent inhibition of the proliferation of both cell lines. This inhibition was correlated with a down-modulation of the cell surface expression of the IL-6 binding chain (gp80) and the transducing chain (gp130) of the IL-6 receptor (IL-6R). Long-term culture experiments showed that down-modulation of gp80 expression was complete at days 15 and 30 in the presence of 10(-5) and 10(-7) mol/l of RA, respectively. Gp 130 expression was greatly decreased, albeit still detectable, in similar culture conditions. RA-mediated interruption of the IL-6 autocrine loop was associated with a decrease of bcl-2 oncoprotein expression and apoptosis of the myeloma cells which was RA concentration- and time-dependent. The in vivo relevance of the effects of RA was studied on tumours which developed in nude mice inoculated with a subclone of RPMI 8226. Whereas tumours grew in all control mice, 40% of tumours regressed within 20 days in RA-treated mice. Cells from regressing tumours featured characteristics of apoptosis and exhibited low gp80 and gp130 expression. Our study indicate that long-term RA treatment interferes in vivo and in vitro with IL-6 autocrine growth of myeloma cell lines, leading to apoptosis.

Topics: Animals; Antigens, CD; Apoptosis; Cell Division; Cell Survival; Cytokine Receptor gp130; Down-Regulation; Growth Substances; Humans; Interleukin-6; Membrane Glycoproteins; Mice; Mice, Nude; Multiple Myeloma; Neoplasm Transplantation; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Receptors, Interleukin; Receptors, Interleukin-6; Transplantation, Heterologous; Tretinoin; Tumor Cells, Cultured

In contrast to cytotoxic agents inducing rapid cell death, biological agents such as hormones, vitamins (e.g., retinoids), cytokines, and antireceptor antibodies act slowly and may alter ratios between cell growth and programmed cell death (apoptosis). We showed previously that anti-interleukin 6 (IL-6) and antitransferrin (Tf) receptor antibodies inhibited in vitro growth and induced death of myeloma cells. Retinoids also inhibit in vitro growth of human cancer cells and decrease IL-6 receptor display and autosecretion by some myeloma cells. Retinoids may also antagonize in vitro growth-promoting effects of iron and transferrin. To develop a novel strategy for treating myeloma, we examined antiproliferative and cytotoxic effects of retinoids in combination with anti-Tf or anti-IL-6 receptor antibodies. Myeloma cell lines were cultured with retinoids with or without anti-growth factor receptor monoclonal antibodies. Both all-trans retinoic acid (ATRA) and 13-cis-retinoic acid showed variable, dose-dependent inhibition of myeloma cell line growth. ATRA also induced significant down-regulation of myeloma IL-6 receptors and inhibited IL-6 autosecretion by myeloma cells. Antiproliferative effects of ATRA were increased by coculture with anti-Tf but not anti-IL-6 receptor antibodies. Colony-forming assays showed that antiproliferative effects of anti-Tf receptor antibodies were largely reversible, but 1 microM ATRA was cytotoxic to myeloma cells. To assess apoptosis, a flow cytometry assay detecting DNA damage was used. Using previously studied cell line models, flow cytometry detected programmed cell death induced by transforming growth factor beta1 in leukemia cells and by anti-growth factor receptor antibody treatment of IL-6-dependent myeloma cells, treatments which caused only modest increases in the percentage of cells undergoing morphological apoptosis and increased internucleosomal DNA degradation. Flow cytometry analysis of ATRA and anti-Tf antibody-treated myeloma cells also showed evidence for apoptosis induced by ATRA, but not with anti-Tf receptor antibodies. These changes were apparent several days before detection of internucleosomal DNA degradation on agarose gels in 8226 cells but were not detected at any time in U266 cells, which underwent cell death but showed no DNA damage using flow cytometry or degradation on agarose gels. Retinoids merit further study as possible maintenance or chemoprevention therapies for clonal plasma cell disorders a

Topics: Antibodies, Monoclonal; Apoptosis; Cell Division; Humans; Multiple Myeloma; Receptors, Interleukin-6; Receptors, Transferrin; Tretinoin; Tumor Cells, Cultured

Topics: Cell Division; Growth Inhibitors; Humans; In Vitro Techniques; Interferon-alpha; Interleukin-6; Multiple Myeloma; Receptors, Interleukin; Receptors, Interleukin-6; Tretinoin; Tumor Cells, Cultured

Retinoic acid has been shown to induce growth inhibition in a variety of cell types including human myeloma cell lines. Bone marrow plasma cells from 31 multiple myeloma (MM) patients were cultured to investigate the activity of 13-cis-retinoic acid (cRA), all-trans-retinoic acid (tRA), interferon-alpha (IFN-alpha), interferon-gamma (IFN-gamma), and dexamethasone (DEX), alone or in combination, on in vitro proliferation and immunoglobulin (Ig) secretion. Both cRA and tRA inhibited proliferation: the labelling index (LI) of treated cultures/controls, was 0.47 +/- 0.05 (mean +/- standard error mean, M +/- SEM) P < 0.0001, and 0.67 +/- 0.04 (M +/- SEM), P < 0.0001, respectively. The inhibitory effect of cRA was significantly superior to tRA (P = 0.0129) and IFN-alpha, similar to IFN-gamma and DEX. The combinations of cRA + IFN alpha, tRA + IFN-gamma, tRA + DEX did not show any synergistic effect on myeloma proliferation. In contrast, the combination cRA + DEX (0.29 +/- 0.04, M +/- SEM) markedly increased the effect of both cRA and DEX used as single agents. Ig synthesis was not significantly affected by CRA, tRA, IFN-gamma and the combination tRA + IFN-gamma. As expected, only IFN-alpha (P = 0.002) and DEX (P < 0.001) inhibited Ig production. The combinations cRA + IFN-alpha, cRA + DEX and tRA + DEX decreased Ig secretion to the same extent as IFN-alpha and DEX alone respectively. In conclusion, our data indicate that tRA and especially cRA strongly inhibited plasma cell proliferation but had no effect on Ig synthesis. The combination of cRA + DEX showed the highest degree of inhibitory activity of all cytokines, alone or in combination.

Topics: Antibodies, Neoplasm; Bromodeoxyuridine; Cell Division; Dexamethasone; Dose-Response Relationship, Drug; Drug Interactions; Humans; Immunoglobulins; Interferon Type I; Interferon-gamma; Isotretinoin; Multiple Myeloma; Recombinant Proteins; Tretinoin; Tumor Cells, Cultured

Topics: Administration, Oral; Aged; Female; Humans; Male; Middle Aged; Multiple Myeloma; Pilot Projects; Tretinoin

Topics: Antineoplastic Agents; Drug Administration Schedule; Drug Resistance; Humans; Leukemia; Leukemia, Promyelocytic, Acute; Leukocytosis; Multiple Myeloma; Tretinoin

We showed the dose-dependent growth inhibition by alltrans retinoic acid (ATRA) of myeloma cells freshly isolated from patients. ATRA downregulated the cell surface expression of interleukin-6 receptor (IL-6R) and/or glycoprotein (gp) 130. The growth-inhibitory activity of ATRA was well correlated with that of anti-gp 130 antibody in every sample. Furthermore, ATRA inhibited the production of IL-6 from both myeloma cells and marrow stromal cells, and recombinant IL-6 (rIL-6) could partially recover the myeloma cell growth that had been inhibited by ATRA. These data suggest that ATRA may inhibit the proliferation of myeloma cells both by the downregulation of IL-6R and gp130 expression on myeloma cells and by the inhibition of IL-6 production from myeloma and stromal cells. Prednisolone (PSL) and interferon-gamma (IFN-gamma) also inhibited the myeloma growth, while their effects were different from those of ATRA on IL-6 R and gp130 expression, IL-6 production, and morphological change. The inhibitory effect of ATRA on myeloma cell proliferation was observed in 10 of 14 samples obtained from eight patients, which suggests that ATRA may be a potent new therapeutic agent for some myeloma patients.

Topics: Antigens, CD; Cell Division; Cytokine Receptor gp130; Down-Regulation; Growth Inhibitors; Humans; Interleukin-6; Membrane Glycoproteins; Multiple Myeloma; Receptors, Interleukin; Receptors, Interleukin-6; Retinaldehyde; Signal Transduction; Tretinoin; Tumor Cells, Cultured; Vitamin A

The addition of lipid hydroperoxides greatly accelerates the rate of oxidative catabolism of all-trans-retinoic acid (RA) in human cell microsomes; hydroperoxy metabolites of the arachidonate cascade are particularly active in the microsomal system. We have measured the plasma content of lipid peroxides in cancer patients during the course of therapy with RA, seeking to assess whether a correlation exists between the rate of oxidative catabolism of exogenously administered RA and whole body lipid peroxide levels. The assay used for plasma lipid peroxides is the capacity to react with thiobarbituric acid under specified conditions; the result is expressed as TBARS (thiobarbituric acid reactive substances). RA administration produced its own accelerated clearance RA within 72 h. Patients were considered to have "normal" or "rapid" baseline catabolism of RA if their Day 1 area under RA concentration over time curve was greater or less than 300 ng.h/ml, respectively. The mean plasma TBARS levels were: 12 normal volunteers = 0.14 microM; 19 "normal" RA catabolizers = 0.25 microM; and 14 "rapid" catabolizers = 0.82 microM. P = 0.008 (rapid catabolizers versus normal volunteers) and 0.05 (rapid catabolizers versus normal catabolizers). Repeat TBARS determinations were made during the course of therapy in 17 patients, all of whom converted to "rapid" RA catabolism on therapy. An increase in plasma TBARS levels > or = 20% of baseline was observed in 5 of 5 prostate cancer patients and 8 of 12 lung cancer patients treated with continuous RA therapy for 2 and 4 weeks, respectively. These observations support the hypothesis that high levels of lipid peroxides and rapid oxidative catabolism of RA are positively correlated.

Topics: Carcinoma, Non-Small-Cell Lung; Humans; Leukemia, Promyelocytic, Acute; Lipid Peroxides; Lung Neoplasms; Male; Metabolic Clearance Rate; Multiple Myeloma; Neoplasms; Prostatic Neoplasms; Reference Values; Thiobarbituric Acid Reactive Substances; Tretinoin

In this report we demonstrate that retinoic acid (RA) down-regulated the number of IL-6R on human leukocyte cell lines, including the myeloma cell line AF10, and two B cell hybridomas that correspond to cells at earlier stages of B cell development. Using AF10 cells, whose growth was determined to be mediated by the autocrine action of IL-6, we found that RA reduction of IL-6R was concentration-dependent over a range of 10(-11) to 10(-5) M and corresponded to the ability of the retinoid to inhibit cell proliferation. The down-regulation of IL-6R number by RA was accompanied by reduced IL-6R mRNA expression. RA did not affect endogeneous IL-6 synthesis or secretion from AF10 cells. However, addition of exogenous rIL-6 could overcome RA-induced growth inhibition. Menthol, a structurally unrelated compound to RA, also suppressed IL-6R expression and, correspondingly, inhibited cell growth. Taken together, our results suggest that the antiproliferative action of RA on AF10 cells is caused by reduction of IL-6R expression and subsequent inhibition of IL-6-mediated autocrine growth. These findings suggest the possibility that down-regulation of IL-6R is a means by which RA can modulate immune function.

Topics: Cell Division; Down-Regulation; Humans; Interleukin-6; Menthol; Multiple Myeloma; Receptors, Immunologic; Receptors, Interleukin-6; Tretinoin; Tumor Cells, Cultured

Necessary for growth and differentiation in many normal tissues and capable of inducing differentiation in human promyelocytic cell lines, retinoids were the subject of this study. Specifically, effects of 13-cis-retinoic acid and 13-trans-retinoic acid on the growth of normal human bone marrow cells in soft-agar system were studied. Both short-term incubation and continuous exposure to retinoic acid caused a decreased number of granulocyte colonies and an increased cluster-to-colony ratio. This effect was concentration-dependent. Examination of specimens stained with Wright-Giemsa or nitro blue tetrazolium stains showed a progressive increase in the percentage of immature granulocytic precursors with increasing concentrations of retinoic acid. No effect of retinoic acid was seen on a number of human tumor cell lines. Retinoic acid blocked both differentiation and proliferation and appeared to do so by specific, noncytotoxic mechanisms in normal human bone marrow cells.

Topics: Bone Marrow; Bone Marrow Cells; Burkitt Lymphoma; Cell Division; Cell Line; Clone Cells; Colony-Forming Units Assay; Colony-Stimulating Factors; Dose-Response Relationship, Drug; Drug Evaluation, Preclinical; Humans; Leukemia, Myeloid; Melanoma; Multiple Myeloma; Osteosarcoma; Tretinoin

The ability of retinoic acid (RA), a potent antitumor agent, to stimulate cell-mediated cytotoxicity (CMC) in mice was investigated. Low doses of RA (5-300 micrograms/mouse/day) administered ip into C57BL/6 mice for 5 days daily or for 1--3 months three times a week before immunization in vivo or in vitro with allogeneic BALB/c S194 myeloma cells led to an enhanced cytotoxic activity of their spleen effector cells. Similarly, in a syngeneic situation injection of RA into C57BL/6 or BALB/c mice before in vitro challenge with EL 4 (C57BL/6) or S194 (BALB/c) tumor cells strongly stimulated CMC. The enhanced cytotoxic activity was effected by thymus-derived lymphocytes (T-cells) and specific for the H-2 histocompatibility antigens in the case of the allogeneic sensitization or specific for tumor antigens in the case of the syngeneic sensitization. Because RA had no effect on the effector step of CMC, RA likely enhanced the induction step of T-CMC. The action of RA was antigen-dependent, and it is therefore a true adjuvant rather than a nonspecific stimulator or polyclonal activator of cytotoxic T-cells.

Topics: Animals; Antibody-Dependent Cell Cytotoxicity; Antigens, Neoplasm; Killer Cells, Natural; Mice; Mice, Inbred Strains; Multiple Myeloma; Neoplasms, Experimental; T-Lymphocytes; Tretinoin; Vitamin A

Topics: Animals; Cytotoxicity, Immunologic; Immunity, Cellular; Mice; Mice, Inbred C57BL; Multiple Myeloma; Neoplasms, Experimental; Tretinoin; Vitamin A