Md. Zahidunnabi Dewan

A novel NF‐κB inhibitor DHMEQ selectively targets constitutive NF‐κB activity and induces apoptosis of multiple myeloma cells in vitro and in vivo

Multiple myeloma (MM) is a fatal lymphoid malignancy that is incurable with conventional modalities of chemotherapy. Strong and constitutive activation of nuclear factor kappa B (NF‐κB) is a common characteristic of MM cells. In our study we successfully target NF‐κB with a novel NF‐κB inhibitor dehydroxymethylepoxyquinomycin (DHMEQ). DHMEQ completely abrogates constitutive NF‐κB activity and induces apoptosis of MM cells, whereas control peripheral blood mononuclear cells (PBMC) are resistant to NF‐κB inhibition and apoptosis by DHMEQ treatment. DHMEQ inhibition of NF‐κB triggers activation of caspases 8 and 9, as well as G0/G1 cell cycle arrest accompanied by downregulation of antiapoptotic genes Bcl‐XL and c‐FLIP and cell cycle progression gene cyclins D1 and D2. DHMEQ‐mediated inhibition of vascular endothelial growth factor (VEGF) production in MM cells raises the possibility that DHMEQ abrogates the autocrine VEGF loop and enhances its antitumor effects by inhibiting neovascularization in the bone marrow. Using an in vivo NOD/SCID/γcnull (NOG) mice model, we show that DHMEQ has a potent inhibitory effect on the growth of MM cells. Compared to other compounds having the potential to inhibit NF‐κB, DHMEQ is a unique compound that blocks the translocation of NF‐κB p65 into the nucleus and selectively targets NF‐κB activated in tumor cells. Therefore, our study presents a new molecular target therapy in MM.

Statin-induced inhibition of HIV-1 release from latently infected U1 cells reveals a critical role for protein prenylation in HIV-1 replication.

Latent infection of human immunodeficiency virus type 1 (HIV-1) represents a major hurdle in the treatment of acquired immunodeficiency syndrome (AIDS) patients. Statins were recently reported to suppress acute HIV-1 infection and reduce infectious virion production, but the precise mechanism of inhibition has remained elusive. Here we demonstrate that lypophilic statins suppress HIV-1 virion release from tumor necrosis factor alpha-stimulated latently infected U1 cells through inhibition of protein geranylgeranylation, but not by cholesterol depletion. Indeed, this suppression was reversed by the addition of geranylgeranylpyrophosphate, and a geranylgeranyltransferase-1 inhibitor reduced HIV-1 production. Notably, silencing of the endogenous Rab11a GTPase expression in U1 cells by RNA interference destabilized Gag and reduced virion production both in vitro and in NOD/SCID/gammac null mice. Our findings thus suggest that small GTPase proteins play an important role in HIV-1 replication, and therefore could be attractive molecular targets for anti-HIV-1 therapy.

Role of natural killer cells in hormone-independent rapid tumor formation and spontaneous metastasis of breast cancer cells in vivo

Natural killer (NK) cells play a central role in host defense against tumor and virus-infected cells. Direct role of NK cells in tumor growth and metastasis remains to be elucidated. We here demonstrated that NOD/SCID/γcnull (NOG) mice lacking T, B and NK cells inoculated with breast cancer cells were efficient in the formation of a large tumor and spontaneous organ-metastasis. In contrast, breast cancer cells produced a small tumor at inoculated site in T and B cell knock-out NOD/SCID mice with NK cells while completely failed to metastasize into various organs. Immunosupression of NOD/SCID by treatment with an anti-murine TM-β1 antibody, which transiently abrogates NK cell activity in vivo, resulted in enhancing tumor formation and organ-metastasis in comparison with non-treated NOD/SCID mice. Activated NK cells inhibited tumor growth in vivo. The rapid and efficient engraftment of the breast cancer cells in NOG mice suggests that this new animal model could provide a unique opportunity to understand and investigate the mechanism of tumor cell growth and metastasis. Our results suggest that NK cells play an important role in cancer growth and metastasis and could be a promising immunotherapeutic strategy against cancer either alone or in combination with conventional therapy.

Natural killer cells in breast cancer cell growth and metastasis in SCID mice

Natural killer (NK) cell is an important component of the innate immune system and plays a central role in host defense against tumor and virus-infected cells. This review briefly summarizes the role of murine NK cells in tumor growth and metastasis of breast cancer cells in severe combined immunodeficiency (SCID) mice. Conventional SCID and NOD-SCID strains have been used to study for xenotransplantion of human tumors. SCID mice models of cancer mimic human diseases and have provided valuable information. However, these mice strains have some residual immunity such as NK cells that somewhat limit post-transplantation growth and metastasis of human xenografts. In contrast, NOD/SCID/gammac(null) (NOG) mice without common gamma-chain inoculated with breast cancer cells were most efficient in the formation of a large tumor and metastasis. NOG mouse strain without NK activity appears to be more promising as tool for xenotransplantion of human cancer. This new xenotransplant model is relevant and can be recommended for use in clarifying the mechanism of growth of cancer cells as well as for developing new therapeutic strategies against cancer.

Natural killer activity of peripheral-blood mononuclear cells in breast cancer patients

Natural killer (NK) activity of immune cells plays a central role in host defense against cancer and virus-infected cells. Natural cytotoxic activity of peripheral-blood mononuclear cells was assessed by a Calcein-AM release assay in 89 subjects. In the present study, we here demonstrated that NK activities of peripheral-blood mononuclear cells (PBMCs) from breast cancer patients were significantly lower as compared with that of healthy individuals. There were significant differences in the NK activities of PBMCs from HER2-negative breast cancer patients as compared with HER2-positive patients. Our results suggest that NK activity of PBMCs is lower in breast cancer indicating a role for immunological natural host defense mechanisms against cancer.

An HIV protease inhibitor, ritonavir targets the nuclear factor-kappaB and inhibits the tumor growth and infiltration of EBV-positive lymphoblastoid B cells

Epstein‐Barr Virus (EBV)‐associated immunoblastic lymphoma occurs in immunocompromised patients such as those with AIDS or transplant recipients after primary EBV infection or reactivation of a preexisting latent EBV infection. In the present study, we evaluated the effect of ritonavir, an HIV protease inhibitor, on EBV‐positive lymphoblastoid B cells in vitro and in mice model. We found that it induced cell‐cycle arrest at G1‐phase and apoptosis through down‐regulation of cell‐cycle gene cyclin D2 and antiapoptotic gene survivin. Furthermore, ritonavir suppressed transcriptional activation of NF‐κB in these cells. Ritonavir efficiently prevented growth and infiltration of lymphoma cells in various organs of NOD/SCID/γcnull mice at the same dose used for treatment of patients with AIDS. Our results indicate that ritonavir targets NF‐κB activated in tumor cells and shows anti‐tumor effects. These data also suggest that this compound may have promise for treatment or prevention of EBV‐associated lymphoproliferative diseases that occur in immunocompromised patients.

Prompt tumor formation and maintenance of constitutive NF-κB activity of multiple myeloma cells in NOD/SCID/γCnull mice

Clinically and biologically relevant animal models are indispensable to evaluate both the pathophysiology and strategies for diagnosis and treatment of multiple myeloma (MM). We examined the tumorigenicity of MM cell lines KMM‐1 and U‐266 in an in vivo cell proliferation model using NOD/SCID/γCnull (NOG) mice. Two cell lines were inoculated either subcutaneously (s.c.) in the post‐auricular region or intravenously (i.v.) in the tail of NOG mice. The KMM‐1 cell line produced a progressively growing large tumor with infiltration of the cells expressing human λ‐chain in various organs of all NOG mice, while the U‐266 cell line failed to do so. Tumor cells grown in NOG mice maintained the original histomorphology, as well as expression patterns of tumor markers human λ, Ig light chain and VEGF. Tumor progression in mice also correlated with elevation of serum human soluble IL‐6R and gp130. Tumor cells sustained a strong NF‐κB activity in vivo and induced NF‐κB components were indistinguishable from those in cells cultured in vitro. The rapid and efficient engraftment of the MM cell line in NOG mice suggests that this is a very useful animal model which could provide a novel system in which to clarify the mechanism of growth of cancer cells, as well as to develop new therapeutic regimens against MM.

Efficient inhibition of SDF‐1α‐mediated chemotaxis and HIV‐1 infection by novel CXCR4 antagonists

CXC chemokine receptor‐4, the receptor for stromal cell‐derived factor‐1α as well as human immunodeficiency virus type 1, belongs to the chemokine receptor family and has been shown to play a critical role in directing the migration of cancer cells to sites of metastasis as well as human immunodeficiency virus type 1 infection. We had previously reported that a duodenally absorbable CXC chemokine receptor‐4 antagonist, KRH‐1636, showed a potent anti‐human immunodeficiency virus type 1 activity both in vivo and in vitro. In this study, we initially examined the effect of the compound and its derivatives on stromal cell‐derived factor‐1α‐mediated chemotaxis of cancer cells in order to evaluate if they could be applicable as a novel inhibitor of cancer metastasis. We found that both KRH‐2731 and KRH‐3955 were highly potent antagonists of stromal cell‐derived factor‐1α‐mediated chemotaxis, i.e. the derivatives exhibited 50% effective concentrations of less than 10 nM, for more than 1000‐fold efficacy improvement over the prototype KRH‐1636. We further demonstrated the greater anti‐human immunodeficiency virus type 1 efficacy of the derivatives compared with the original KRH‐1636. Taken together, the KRH‐1636 derivatives KRH‐2731 and KRH‐3955 may be promising as a novel inhibitory drug for cancer metastasis as well as for human immunodeficiency virus type 1 infection. (Cancer Sci 2009; 100: 778ndash;781)

Hodgkin's lymphoma cells are efficiently engrafted and tumor marker CD30 is expressed with constitutive nuclear factor-κB activity in unconditioned NOD/SCID/γcnull mice

As there are very few reproducible animal models without conditioning available for the study of human B‐cell‐type Hodgkins lymphoma (HL), we investigated the ability of HL cells to induce tumors using novel NOD/SCID/γcnull (NOG) mice. Four human Epstein–Barr virus‐negative cell lines (KM‐H2 and L428 originated from B cells, L540 and HDLM2 originated from T cells) were inoculated either subcutaneously in the postauricular region or intravenously in the tail of unmanipulated NOG mice. All cell lines successfully engrafted and produced tumors with infiltration of cells in various organs of all mice. Tumor cells had classical histomorphology as well as expression patterns of the tumor marker CD30, which is a cell surface antigen expressed on HL. Tumor progression in mice inoculated with B‐cell‐type, but not T‐cell‐type, HL cells correlated with an elevation in serum human interleukin‐6 levels. Tumor cells from the mice also retained strong nuclear factor (NF)‐κB DNA binding activity, and the induced NF‐κB components were indistinguishable from those cultured in vitro. The reproducible growth behavior and preservation of characteristic features of both B‐cell‐type and T‐cell‐type HL in the mice suggest that this new xenotransplant model can provide a unique opportunity to understand and investigate the mechanism of pathogenesis and malignant cell growth, and to develop novel anticancer therapies. (Cancer Sci 2005; 96: 466–473)

Induction of apoptosis in Epstein-Barr virus-infected B-lymphocytes by the NF-κB inhibitor DHMEQ

Epstein-Barr virus (EBV) causes EBV-associated lymphoproliferative diseases in patients with profound immune suppression. Most of these diseases are life-threatening and the prognosis of AIDS-associated lymphomas is extremely unfavorable. Polyclonal expansion of virus infected B-cell predisposes them to transformation. We investigated the possibility of nuclear factor kappa B (NF-kappaB) inhibition by dehydroxymethylepoxyquinomicin (DHMEQ) for the treatment and prevention of EBV-associated lymphoproliferative diseases. We examined the effect of DHMEQ on apoptosis induction in four EBV-transformed lymphoblastoid cell lines as well as peripheral blood mononuclear cells infected with EBV under immunosuppressed condition. DHMEQ inhibits NF-kappaB activation in EBV-transformed lymphoblastoid cell lines and induces apoptosis by activation of mitochondrial and membranous pathways. Using an in vivo NOD/SCIDgammac mouse model, we showed that DHMEQ has a potent inhibitory effect on the growth of lymphoblastoid cells. In addition, DHMEQ selectively purges EBV-infected cells expressing latent membrane protein (LMP) 1 from peripheral blood mononuclear cells and inhibits the outgrowth of lymphoblastoid cells. These results suggest that NF-kappaB is a molecular target for the treatment and prevention of EBV-associated lymphoproliferative diseases. As a potent NF-kappaB inhibitor, DHMEQ is a potential compound for applying this strategy in clinical medicine.