Interferon-gamma sensitizes hepatitis B virus-expressing hepatocarcinoma cells to 5-fluorouracil through inhibition of hepatitis B virus-mediated nuclear factor-kappaB activation

Nuclear factor (NF)-κB is important for immune responses and cell survival; however, abnormal activation of NF-κB is linked with many types of diseases, including hepatocellular carcinoma (HCC). Our previous report indicated that hepatitis B virus (HBV) induces NF-κB activation through NF-κB-inducing kinase (NIK), and this can be blocked specifically by interferon (IFN)-γ. In the present study, we report that HBV expression in HCC cell lines induces drug resistance against 5-fluorouracil (5-FU). This drug resistance was abolished by inhibition of NF-κB activation through small interfering RNA-mediated NIK 'knockdown' and IFN-γ treatment. In addition to the reduced NF-κB activation and drug resistance, the upregulated growth arrest- and DNA damage-inducible protein 45β (Gadd45β) in HBV-expressing HCC cell lines was downregulated by the small interfering RNA-mediated NIK knockdown and IFN-γ treatment. The overexpression of Gadd45β in HCC cell lines also induces drug resistance against 5-FU. Based on our data, we suggest that IFN-γ treatment might be helpful for chemotherapy in HBV-integrated HCC through inhibition of the NIK-mediated NF-κB activation and downregulation of the NF-κB target gene Gadd45β . ( Cancer Sci 2007; 98: 1758–1766)     

Transient inhibition of NF-κB by DHMEQ induces cell death of primary effusion lymphoma without HHV-8 reactivation

Primary effusion lymphoma (PEL) is a refractory malignancy caused by human herpes virus 8 (HHV-8) in immunocompromised individuals. The tumor cells of PEL are characterized by constitutive NF-κB activation. Dehydroxymethylepoxyquinomicin (DHMEQ) is a new NF-κB inhibitor and is effective on various tumor cells with constitutively activated NF-κB. Thus, in search for a new therapeutic modality of PEL, we examined the effect of DHMEQ on PEL cells. We confirmed constitutive activation of NF-κB with subcomponents of p50 and p65 in PEL cell lines. DHMEQ quickly and transiently abrogated NF-κB activation and reduced the cell viability in dose- and time-dependent manners, inducing apoptosis through activation of both mitochondrial and membrane pathways. Array analysis revealed that DHMEQ down-regulated expression levels of NF-κB target genes, such as interleukin-6 (IL6), Myc , chemokine (C-C motif) receptor 5 ( CCR5 ) and NF-κB1, whereas it up-regulated expression levels of some genes involved in apoptosis, and cell cycle arrest. DHMEQ did not reactivate HHV-8 lytic genes, indicating that NF-κB inhibition by DHMEQ did not induce virus replication. DHEMQ rescued CB-17 SCID mice xenografted with PEL cells, reducing the gross appearance of effusion. Thus, DHMEQ transiently abrogated the NF-κB activation, irreversibly triggering the apoptosis cascade without HHV-8 reactivation. In addition, DHMEQ could rescue the PEL-xenograft mice. Therefore, we suggest DHMEQ as a promising candidate for molecular target therapy of the PEL. ( Cancer Sci 2009; 100: 737–746)     

Nuclear factor-kappaB dependency of doxorubicin sensitivity in gastric cancer cells is determined by manganese superoxide dismutase expression

The role of nuclear factor-κB (NF-κB) activation in cancer cell apoptosis appears to be tailored specifically for each cell type and the type of NF-κB inducer. The present study aimed to determine whether or not NF-κB activation is associated with chemosensitivity to doxorubicin (DOX) using the DOX-sensitive SNU-601 and DOX-resistant SNU-216 gastric cancer cell lines. The effect of NF-κB activation on DOX (1 µg/mL) sensitivity was analyzed after the suppression of NF-κB activation using transfection of the super-suppressive mutant form of IκBα (mIκBα) or pretreatment with pyrrolidine dithiocarbamate. In addition, the association between NF-κB and manganese superoxide dismutase (MnSOD) in relation to DOX sensitivity was analyzed after the modulation of MnSOD expression. The NF-κB activity was much higher in DOX-resistant SNU-216 cells than in DOX-sensitive SNU-601 cells before and after DOX treatment. Overexpression of mIκBα or pyrrolidine dithiocarbamate pretreatment decreased the DOX resistance in SNU-601 cells with low MnSOD expression, but not in SNU-216 cells with high MnSOD expression. In comparison, the overexpression of MnSOD, which also suppressed NF-κB activation in both cell lines, increased DOX resistance in SNU-601 cells. Blocking of MnSOD expression using RNA interference techniques increased DOX sensitivity in SNU-216 cells, which was further augmented by the additional inhibition of NF-κB activity. Our results showed that whether NF-κB contributes to DOX sensitivity in gastric cancer cells is determined by the level of MnSOD expression. Thus, targeting both MnSOD and NF-κB may be helpful for increasing the efficacy of DOX treatment of DOX-resistant SNU gastric cancer cells. ( Cancer Sci 2008; 99: 1117–1124)     

Reduction of human T-cell leukemia virus type-1 infection in mice lacking nuclear factor-kappaB-inducing kinase

Human T-cell lymphotropic virus type 1 (HTLV-1) causes adult T-cell leukemia and inflammatory disorders. Aberrant activation of nuclear factor-κB (NF-κB) has been linked to HTLV-1 pathogenesis and to various kinds of cancers, including adult T-cell leukemia. NF-κB-inducing kinase (NIK) is critical for non-canonical activation of NF-κB and for the development of lymphoid organs. HTLV-1 activates NF-κB by the non-canonical pathway, but examination of the role of NIK in proliferation of HTLV-1-infected cells in vivo has been hindered by lack of a suitable animal model. Alymphoplasia ( aly/aly ) mice bear a mutation of NIK, resulting in defects in the development of lymphoid organs and severe deficiencies in both humoral and cell-mediated immunity. In the present study we therefore used a mouse model of HTLV-1 infection with aly/aly mice. The number of HTLV-1-infected cells in the reservoir organs in aly/aly mice was significantly smaller than in the control group 1 month after infection. In addition, aly/aly mice did not maintain provirus for 1 year and antibodies against HTLV-1 were undetectable. These results demonstrate that the absence of functional NIK impairs primary HTLV-1 proliferation and abolishes the maintenance of provirus. Interestingly, clonal proliferation of HTLV-1-infected mouse cells was not detected in aly/aly mice, which is consistent with the lack of HTLV-1 persistence. These observations imply that the clonal proliferation of HTLV-1-infected cells in secondary lymphoid organs might be important for HTLV-1 persistence. ( Cancer Sci 2008; 99: 872–878)     

Rearranged NF-kappa B2 gene in an adult T-cell leukemia cell line

Adult T-cell leukemia (ATL) is an aggressive type of leukemia, originating from T-cells infected with human T-cell leukemia virus type 1. Accumulating evidence suggests the aberrant activation of NF-κB to be a causative factor mediating the abnormal proliferation of leukemic cells, thus resulting in the development of ATL. A rearranged NF-κB2/p100 gene was isolated from an ATL-derived cell line, which was generated by a chromosomal translocation. The isolated NF-κB2 mutant is fused with the with no (lysine) deficient protein kinase 1 gene, coding for a 58 kDa protein that retains the DNA binding Rel homology domain, but it lacks the entire ankyrin repeat inhibitory domain, thus suggesting its constitutive activation. This rearranged NF-κB2 gene product (p58) was localized in the nucleus, and formed a complex with NF-κB p65 or RelB. Moreover, a T-cell line expressing p58 increased the amount of an NF-κB2-inducible gene, NF-κB2/p100 by itself. These results suggest that such NF-κB2 gene rearrangement may therefore be a factor in the constitutive activation of NF-κB in ATL, and thereby playing a role in the ATL pathogenesis. ( Cancer Sci 2008; 99: 792–798)     

T helper responses are maintained by basal-like breast cancer cells and confer to immune modulation via upregulation of PD-1 ligands

A conspicuous T cell infiltration is frequently observed in triple-negative and/or basal-like breast cancers. Since the immunological course of breast cancer is explicitly directed by helper T cells, this study aims to determine the influence of basal-like breast cancer (BLBC) cells on CD4⁺ T cell responses. Co-cultures were established with breast cancer cell lines and CD4⁺ T cells under stimulatory conditions. Helper T cell activation, proliferation, cytokine secretion, and differentiation were assessed. Protein and mRNA expression of PD-1 ligands were determined on breast cancer cell lines. Blockade assays were performed in order to determine the functional assets of PD-1 ligation. In contrast to luminal breast cancer cells, BLBC cells allowed CD4⁺ T cell activation, proliferation, and IFN-γ secretion, but only to a certain extent. A substantial population of CD25⁺CD127^low/? regulatory T (Treg) cells was also induced in BLBC co-cultures. In return, IFN-γ stimulated the upregulation of PD-L1 (B7-H1) and/or PD-L2 (B7-DC) inhibitory molecules on the basal-like cells. In prolonged periods of co-culturing, blockade of PD-1 ligands on BLBC cell lines impaired Treg differentiation, restored IL-2 secretion, and increased CD8⁺ T cell activation. In conclusion, T helper responses were maintained by BLBC cells. On the other hand, IFN-γ secreted from Th1 and other immune cells upregulated the expression of PD-1 ligands on BLBC cells and modulated the immune reactions. Our results indicate the capacity of BLBCs to adapt to IFN-γ-mediated anti-tumor immune responses and to evade immunity via upregulation of PD-1 ligands.     

Immunohistochemical characterization of subtypes of male breast carcinoma

Introduction  

Male breast cancer accounts for around 1% of all breast cancer cases but the incidence has risen in recent years. This study aimed to classify the molecular subtypes of male breast cancers based on the expression profile of immunomarkers and to evaluate their association with clinicopathological features and expression patterns of epidermal growth factor receptor (EGFR) and nuclear factor κB (NF-κB).     

Vitamin E succinate induced apoptosis and enhanced chemosensitivity to paclitaxel in human bladder cancer cells in vitro and in vivo

There have been several studies on the antitumor activities of vitamin E succinate (α-TOS) as complementary and alternative medicine. In the present study, we investigated the cytotoxic effect of α-TOS and the enhancement of chemosensitivity to paclitaxel by α-TOS in bladder cancer. KU-19-19 and 5637 bladder cancer cell lines were cultured in α-TOS and/or paclitaxel in vitro . Cell viability, flow cytometric analysis, and nuclear factor-kappa B (NF-κB) activity were analyzed. For in vivo therapeutic experiments, pre-established KU-19-19 tumors were treated with α-TOS and/or paclitaxel. In KU-19-19 and 5637 cells, the combination treatment resulted in a significantly higher level of growth inhibition, and apoptosis was significantly induced by the combination treatment. NF-κB was activated by paclitaxel; however, the activation of NF-κB was inhibited by α-TOS. Also, the combination treatment significantly inhibited tumor growth in mice. In the immunostaining of the tumors, apoptosis was induced and proliferation was inhibited by the combination treatment. Combination treatment of α-TOS and paclitaxel showed promising anticancer effects in terms of inhibiting bladder cancer cell growth and viability in vitro and in vivo . One of the potential mechanisms by which the combination therapy has synergistic cytotoxic effects against bladder cancer may be that α-TOS inhibits NF-κB induced by chemotherapeutic agents. ( Cancer Sci 2009;)     

RETRACTED ARTICLE: Platelet-derived growth factor-D contributes to aggressiveness of breast cancer cells by up-regulating Notch and NF-κB signaling pathways

Platelet-derived growth factor-D (PDGF-D) has been linked with several human malignancies; however, its role in breast cancer progression is not known. We found that PDGF-D expressing breast cancer cell lines MDA-MB-231 and SUM-149 are more invasive compared to cell lines with little or no expression of PDGF-D such as MDA-MB-468 and MCF-7 cells. Over-expression of PDGF-D in PDGF-D low expressing MDA-MB-468 and MCF-7 cells by cDNA transfection showed increased cell proliferation while silencing the expression of PDGF-D by siRNA in PDGF-D high expressing MDA-MB-231 and SUM-149 cells showed decreased cell proliferation and increased apoptosis. Moreover, PDGF-D over-expression was positively correlated with the expression of Notch-1 and Jagged-1, and the expression of mesenchymal markers (Vimentin and ZEB-2) with concomitant decreased expression of epithelial marker E-cadherin. Since NF-κB activation plays a crucial role in Notch signaling as well as in epithelial–mesenchymal transition and tumor aggressiveness, we determined the DNA binding activity of NF-κB and our findings are consistent showing that PDGF-D over-expression led to increased DNA binding activity of NF-κB while it was found to be decreased by inactivation of PDGF-D. These results were also consistent with the expression and activity of MMP-9 and VEGF, as well as invasive characteristics. Further, forced expression of Notch-1/Jagged-1 by cDNA transfection de-repressed the effects of PDGF-D silencing on NF-κB activity and invasion. From these results, we conclude that PDGF-D plays an important role in breast tumor aggressiveness and this process is mechanistically linked with the activation of Notch and NF-κB signaling.     

Does the PI3K pathway play a role in basal breast cancer?

Many cell kinases exert their proliferative and pro-survival effects through activation of the phosphatidylinositol 3-kinase (PI3K)-Akt pathway. Basal-like breast cancer is a subtype that can be identified by molecular analysis and often includes tumors lacking expression of estrogen receptor/progesterone receptor or human epidermal growth factor receptor, also known as triple-negative breast cancers. Triple-negative cancers comprise < 20% of all breast cancers and have no obvious mechanism driving proliferation, yet these tumors demonstrate higher levels of Akt activation compared with non-triple-negative breast cancers. This suggests a possible role for targeting the PI3K pathway for the treatment of this subset of aggressive cancers. Most clinical trials which have attempted targeting the PI3K/Akt pathway in triple-negative breast cancer have involved the use of EGFR inhibitors with limited success. Novel agents targeting PI3K are under development in early-phase clinical trials and may demonstrate benefit in combination with chemotherapy or other targeted agents such as mitogen-activated protein kinase inhibitors for the treatment of triple-negative or basal-like breast cancer.     

Breast Cancer Classification to Select Patients for Cancer Treatment

Gene expression arrays have shown that breast cancer is comprised of at least four different molecular diseases. These are 1) basal-like breast cancer, 2) human epidermal growth factor receptor 2 (Her2)-positive/estrogen receptor (ER)-negative breast cancer; 3) luminal A breast cancer; and 4) luminal breast B cancer. Basal-like cancer is characterized by ER-negative, progesterone receptor–negative, and Her2-negative expressions. Luminal breast cancer is characterized by ER-positive expression, with the luminal B subtype exhibiting a higher expression of proliferation genes. Several studies have shown that luminal A breast cancer exhibits a better outcome compared with other classes. Also, recent studies have shown that the same subclass is less chemosensitive. These data suggest that some selected luminal A breast cancer cases could be spared adjuvant chemotherapy given their better outcome and lesser chemosensitivity compared with other subtypes of breast cancer. Although other subtypes are more chemosensitive, there is debate about whether some drugs could be active in specific subtypes. Anthracyclines have been reported to be more effective in Her2-overexpressing breast cancer, and cisplatin is currently under investigation in basal-like breast cancer. Finally, some data suggest that luminal B breast cancer exhibits higher sensitivity to aromatase inhibitors. Overall, molecular classes exhibit different sensitivity to conventional drugs. To what extent these data obtained in retrospective biomarker studies could be implemented in daily practice is a matter of debate given the heterogeneity of findings in different studies.