Sita Aggarwal

Inhibition of growth and survival of human head and neck squamous cell carcinoma cells by curcumin via modulation of nuclear factor-κB signaling

Increased expression of proinflammatory and proangiogenic factors are associated with aggressive tumor growth and decreased survival of patients with head and neck squamous cell carcinoma (HNSCC). In as much as genes that are regulated by nuclear factor NF‐κB suppress apoptosis, induce proliferation, and mediate inflammation, angiogenesis and tumor metastasis, agents that suppress NF‐κB activation have potential as treatment for various cancers including HNSCC. We demonstrate that all HNSCC cell lines expressed constitutively active NF‐κB and IκBα kinase (IKK), which is needed for NF‐κB activation. Treatment of MDA 686LN cells with curcumin (diferuloylmethane), a pharmacologically safe chemopreventive agent, inhibited NF‐κB activation through abrogation of IKK. As a result expression of various cell survival and cell proliferative genes including Bcl‐2, cyclin D1, IL‐6, COX‐2 and MMP‐9 was suppressed. This, in turn, inhibits proliferation of all HNSCC cell lines, arrests cell cycle in G1/S phase (MDA 686LN) and induces apoptosis as indicated by upstream and downstream caspase activation, PARP cleavage, annexin V staining in MDA 686LN cells. Suppression of NF‐κB by cell‐permeable p65‐based peptide and NBD peptide also inhibited the proliferation and induced apoptosis in these cells. Our results indicate that curcumin is a potent inhibitor of cell proliferation and an inducer of apoptosis in HNSCC through suppression of IKK‐mediated NF‐κB activation and of NF‐κB‐regulated gene expression.

Growth suppression of lung cancer cells by targeting cyclic AMP response element-binding protein

Genes regulated by cyclic AMP-response element-binding protein (CREB) have been reported to suppress apoptosis, induce cell proliferation, and mediate inflammation and tumor metastasis. However, it is not clear whether CREB is critically involved in lung carcinogenesis. We found that non-small cell lung cancer (NSCLC) cell lines exhibited elevated constitutive activity in CREB, in its immediate upstream kinases (ribosomal s6 kinase and extracellular signal kinase), and in the CREB-regulated cell survival proteins Bcl-2 and Bcl-xL. We hypothesized that constitutively active CREB is important to lung cancer cell growth and survival and therefore could be a potential therapeutic target for NSCLC. Ectopic expression of dominant repressor CREB and transfection with small interfering RNA against CREB suppressed the growth and survival of NSCLC cells and induced apoptotic cell death. Furthermore, treating H1734 NSCLC cells with an inhibitor of the CREB signaling pathway Ro-31-8220 inhibited CREB activation by blocking the activity of extracellular signal kinase and ribosomal s6 kinase, arrested the cell cycle at the G(2)-M phase, and subsequently induced apoptosis with the suppression of Bcl-2 and Bcl-xL expression. Ro-31-8220 suppressed both the anchorage-dependent and independent growth of NSCLC cells, but its cytotoxic effect was much less prominent in normal bronchial epithelial cells. Our results indicate that active CREB plays an important role in NSCLC cell growth and survival. Thus, agents that suppress CREB activation could have potential therapeutic value for NSCLC treatment.

Melanoma Differentiation-Associated Gene-7/IL-24 Gene Enhances NF-κB Activation and Suppresses Apoptosis Induced by TNF

Melanoma differentiation-associated gene-7 (mda-7), also referred to as IL-24, is a novel growth regulatory cytokine that has been shown to regulate the immune system by inducing the expression of inflammatory cytokines, such as TNF, IL-1, and IL-6. Whether the induction of these cytokines by MDA-7 is mediated through activation of NF-κB or whether it regulates cytokine signaling is not known. In the present report we investigated the effect of MDA-7 on NF-κB activation and on TNF-induced NF-κB activation and apoptosis in human embryonic kidney 293 cells. Stable or transient transfection with mda-7 into 293 cells failed to activate NF-κB. However, TNF-induced NF-κB activation was significantly enhanced in mda-7-transfected cells, as indicated by DNA binding, p65 translocation, and NF-κB-dependent reporter gene expression. Mda-7 transfection also potentiated NF-κB reporter activation induced by TNF receptor-associated death domain and TNF receptor-associated factor-2. Cytoplasmic MDA-7 with deleted signal sequence was as effective as full-length MDA-7 in potentiating TNF-induced NF-κB reporter activity. Secretion of MDA-7 was not required for the potentiation of TNF-induced NF-κB activation. TNF-induced expression of the NF-κB-regulated gene products cyclin D1 and cyclooxygenase-2, were significantly up-regulated by stable expression of MDA-7. Furthermore, MDA-7 expression abolished TNF-induced apoptosis, and suppression of NF-κB by IκBα kinase inhibitors enhanced apoptosis. Overall, our results indicate that stable or transient MDA-7 expression alone does not substantially activate NF-κB, but potentiates TNF-induced NF-κB activation and NF-κB-regulated gene expression. Potentiation of NF-κB survival signaling by MDA-7 inhibits TNF-mediated apoptosis.

Mouse Apolipoprotein B Editing Complex 3 (APOBEC3) Is Expressed in Germ Cells and Interacts with Dead-End (DND1)

Background The dead-end (Dnd1) gene is essential for maintaining the viability of germ cells. Inactivation of Dnd1 results in sterility and testicular tumors. The Dnd1 encoded protein, DND1, is able to bind to the 3′-untranslated region (UTR) of messenger RNAs (mRNAs) to displace micro-RNA (miRNA) interaction with mRNA. Thus, one function of DND1 is to prevent miRNA mediated repression of mRNA. We report that DND1 interacts specifically with APOBEC3. APOBEC3 is a multi-functional protein. It inhibits retroviral replication. In addition, recent studies show that APOBEC3 interacts with cellular RNA-binding proteins and to mRNA to inhibit miRNA-mediated repression of mRNA. Methodology/Principal Findings Here we show that DND1 specifically interacts with another cellular protein, APOBEC3. We present our data which shows that DND1 co-immunoprecipitates APOBEC3 from mammalian cells and also endogenous APOBEC3 from mouse gonads. Whether the two proteins interact directly remains to be elucidated. We show that both DND1 and APOBEC3 are expressed in germ cells and in the early gonads of mouse embryo. Expression of fluorescently-tagged DND1 and APOBEC3 indicate they localize to the cytoplasm and when DND1 and APOBEC3 are expressed together in cells, they sequester near peri-nuclear sites. Conclusions/Significance The 3′-UTR of mRNAs generally encode multiple miRNA binding sites as well as binding sites for a variety of RNA binding proteins. In light of our findings of DND1-APOBEC3 interaction and taking into consideration reports that DND1 and APOBEC3 bind to mRNA to inhibit miRNA mediated repression, our studies implicate a possible role of DND1-APOBEC3 interaction in modulating miRNA-mediated mRNA repression. The interaction of DND1 and APOBEC3 could be one mechanism for maintaining viability of germ cells and for preventing germ cell tumor development.

Erratum: Inhibition of growth and survival of human head and neck squamous cell carcinoma cells by curcumin via modulation of nuclear factor-κB signalling (International Journal of Cancer (2004) 111 (679-692))

Due to the investigators being supplied the wrong antibody by the manufacturer, the reported detection of human hairy and enhancer of split-1 (HES1) protein in Merkel cell carcinoma cell lines is incorrect. The protein detected was human embryonic stem cell 1 (HES1); hence Figure 5 should be ignored. Similarly, the primers used for the RT-PCR were for the gene encoding human embryonic stem cell 1, not human hairy and enhancer of split-1; hence panel 4 of Figure 3a should be ignored. We are highly fortunate that the results we obtained do not affect the conclusions of our paper, which are that Brn-3c and HATH1 are expressed in normal Merkel cells and in ‘classic’ Merkel cell carcinoma lines but not in ‘variant’ Merkel cell carcinoma lines. However, there are two minor passages of text in our manuscript that require correction. On the bottom of page 106 ignore the text “HES1 transcripts were detected in all cell lines examined (Figure 3a, Panel 4), but as RNA levels may not reflect protein levels, HES1 protein levels were determined by western analysis. Examination of protein lysates indicated the 28 kDa isoform of HES1 was present in all cell lines examined (MCC, SCLC and Ewing’s sarcoma) in approximately equal quantities (Figure 5), and thus is unlikely to be responsible for the lack of NE phenotype in MCC adherent cultures.” On page 109, final paragraph, ignore the text “and our results suggest HES1 may not be responsible for the decrease in NE expression in Variant cell lines.”

Abstract 5600: Destruction of prostate cancer cell xenografts by FSH-Lytic peptide conjugates.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Background: In previous studies (Hansel, et al., Mol. and Cell. Endocrinol. 269:26-33, 2007),we showed that conjugates of membrane destroying lytic peptides with either LHRH or with a 15-amino acid segment of the β chain of human chorionic gonadotropin (hCG) target and destroy human prostate, breast and ovarian cancer cells in tumor bearing nude mice. Recently (Radu et al., N. England J. Med. 363:1621-1630, 2012) reported that endothelial cells in blood vessels supplying cancers express follicle-stimulating hormone (FSH) receptors. Objectives:The objectives of this study were to synthesize a bioconjugate of a lyticpeptide (Phor18) to each of three segments of the β chain of FSH that are known to bind to the FSH receptor, and test these conjugates (FSH90-95-Phor18, FSH81-95-Phor18 and FSH33-53-Phor18) for their ability to target and lyse prostate cancer cells in vitro and in vivo. Results: In in vivo experiments, administration of FSH90-95-Phor18 and FSH81-95-Phor18 significantly (p 0.05). The average tumor volume was maintained at significantly lower levels in the mice treated with FSH90-95-Phor18 (p=0.027), FSH81-95-Phor18 (p=0.029) when compared with vehicle and free peptide treatment groups during the study (p < 0.05). Immunohistochemical analyses for FSHR were performed on the tumor samples. FSHR-positive endothelial cells were found in many vessels supplying the tumors of control mice, but few FSHR-positive endothelial cells were present in tumors of mice treated with FSH90-95-Phor18, FSH81-95-Phor 18 or FSH33-53-Phor 18. FSH81-95-Phor 18 was the most effective of the three conjugates tested in destroying the FSHR-bearing endothelial cells and in inhibiting tumor growth of the PC-3 xenografts in nude mice. Conclusion: These data show that Phor18 conjugates of FSH β chain segments that bind to FSHR expressed by the endothelial cells of the blood vessels supplying the tumors are capable of inhibiting prostate cancer cell tumor growth by inhibiting angiogenesis. These FSH β chain-lytic peptide conjugates may be useful in treating prostate and other FSHR expressing cancers. Citation Format: Sita Aggarwal, Ted Gauthier, Hector Alila, Carola Leuschner, Namrata Karki, Rajasree Solipuram, William Hansel. Destruction of prostate cancer cell xenografts by FSH-Lytic peptide conjugates. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5600. doi:10.1158/1538-7445.AM2013-5600