Alok C. Bharti

Nuclear factor-kappa B and cancer: its role in prevention and therapy

Cancer is a hyperproliferative disorder in which invasion and angiogenesis lead to tumor metastasis. Several genes that mediate tumorigenesis and metastasis are regulated by a nuclear transcription factor, nuclear factor kappa B (NF-kappaB). A heterotrimeric complex consisting of p50, p65, and IkappaBalpha, NF-kappaB is present in its inactive state in the cytoplasm. When NF-kappaB is activated, IkappaBalpha is degraded and p50-p65 heterodimer is translocated to the nucleus, binds the DNA (at the promoter region), and activates gene. Research within the last few years has revealed that NF-kappaB is activated by carcinogens, tumor promoters, inflammatory cytokines, and by chemotherapeutic agents. The activation of NF-kappaB can suppress apoptosis, thus promoting chemoresistance and tumorigenesis. Interestingly, however, most chemopreventive agents appear to suppress the activation of the NF-kappaB through inhibition of NF-kappaB signaling pathway. These chemopreventive agents also sensitize the tumors to chemotherapeutic agents through abrogation of NF-kappaB activation. Overall, these observations suggest that NF-kappaB is an ideal target for chemoprevention and chemosensitization. This article reviews evidence supporting this hypothesis.

Curcumin (Diferuloylmethane) Inhibits Constitutive and IL-6-Inducible STAT3 Phosphorylation in Human Multiple Myeloma Cells

Numerous reports suggest that IL-6 promotes survival and proliferation of multiple myeloma (MM) cells through the phosphorylation of a cell signaling protein, STAT3. Thus, agents that suppress STAT3 phosphorylation have potential for the treatment of MM. In the present report, we demonstrate that curcumin (diferuloylmethane), a pharmacologically safe agent in humans, inhibited IL-6–induced STAT3 phosphorylation and consequent STAT3 nuclear translocation. Curcumin had no effect on STAT5 phosphorylation, but inhibited the IFN-α-induced STAT1 phosphorylation. The constitutive phosphorylation of STAT3 found in certain MM cells was also abrogated by treatment with curcumin. Curcumin-induced inhibition of STAT3 phosphorylation was reversible. Compared with AG490, a well-characterized Janus kinase 2 inhibitor, curcumin was a more rapid (30 min vs 8 h) and more potent (10 μM vs 100 μM) inhibitor of STAT3 phosphorylation. In a similar manner, the dose of curcumin completely suppressed proliferation of MM cells; the same dose of AG490 had no effect. In contrast, a cell-permeable STAT3 inhibitor peptide that can inhibit the STAT3 phosphorylation mediated by Src blocked the constitutive phosphorylation of STAT3 and also suppressed the growth of myeloma cells. TNF-α and lymphotoxin also induced the proliferation of MM cells, but through a mechanism independent of STAT3 phosphorylation. In addition, dexamethasone-resistant MM cells were found to be sensitive to curcumin. Overall, our results demonstrated that curcumin was a potent inhibitor of STAT3 phosphorylation, and this plays a role in the suppression of MM proliferation.

Piceatannol Inhibits TNF-Induced NF-κB Activation and NF-κB-Mediated Gene Expression Through Suppression of IκBα Kinase and p65 Phosphorylation

Piceatannol is an anti-inflammatory, immunomodulatory, and anti-proliferative stilbene that has been shown to interfere with the cytokine signaling pathway. Previously, we have shown that resveratrol suppresses the activation of the nuclear transcription factor NF-κB. Piceatannol, previously reported as a selective inhibitor of protein tyrosine kinase Syk, is structurally homologous to resveratrol. Whether piceatannol can also suppress NF-κB activation was investigated. The treatment of human myeloid cells with piceatannol suppressed TNF-induced DNA binding activity of NF-κB. In contrast, stilbene or rhaponticin (another analog of piceatannol) had no effect, suggesting the critical role of hydroxyl groups. The effect of piceatannol was not restricted to myeloid cells, as TNF-induced NF-κB activation was also suppressed in lymphocyte and epithelial cells. Piceatannol also inhibited NF-κB activated by H2O2, PMA, LPS, okadaic acid, and ceramide. Piceatannol abrogated the expression of TNF-induced NF-κB-dependent reporter gene and of matrix metalloprotease-9, cyclooxygenase-2, and cyclin D1. When examined for the mechanism, we found that piceatannol inhibited TNF-induced IκBα phosphorylation, p65 phosphorylation, p65 nuclear translocation, and IκBα kinase activation, but had no significant effect on IκBα degradation. Piceatannol inhibited NF-κB in cells with deleted Syk, indicating the lack of involvement of this kinase. Overall, our results clearly demonstrate that hydroxyl groups of stilbenes are critical and that piceatannol, a tetrahydroxystilbene, suppresses NF-κB activation induced by various inflammatory agents through inhibition of IκBα kinase and p65 phosphorylation.

(DIFERULOYLMETHANE) INHIBITS RECEPTOR ACTIVATOR OF NFKAPPA B LIGAND-INDUCED NF-KAPPA B ACTIVATION IN OSTEOCLAST PRECURSORS AND SUPPRESSES OSTEOCLASTOGENESIS

Numerous studies have indicated that inflammatory cytokines play a major role in osteoclastogenesis, leading to the bone resorption that is frequently associated with cancers and other diseases. Gene deletion studies have shown that receptor activator of NF-κB ligand (RANKL) is one of the critical mediators of osteoclastogenesis. How RANKL mediates osteoclastogenesis is not fully understood, but an agent that suppresses RANKL signaling has potential to inhibit osteoclastogenesis. In this report, we examine the ability of curcumin (diferuloylmethane), a pigment derived from turmeric, to suppress RANKL signaling and osteoclastogenesis in RAW 264.7 cells, a murine monocytic cell line. Treatment of these cells with RANKL activated NF-κB, and preexposure of the cells to curcumin completely suppressed RANKL-induced NF-κB activation. Curcumin inhibited the pathway leading from activation of IκBα kinase and IκBα phosphorylation to IκBα degradation. RANKL induced osteoclastogenesis in these monocytic cells, and curcumin inhibited both RANKL- and TNF-induced osteoclastogenesis and pit formation. Curcumin suppressed osteoclastogenesis maximally when added together with RANKL and minimally when it was added 2 days after RANKL. Whether curcumin inhibits RANKL-induced osteoclastogenesis through suppression of NF-κB was also confirmed independently, as RANKL failed to activate NF-κB in cells stably transfected with a dominant-negative form of IκBα and concurrently failed to induce osteoclastogenesis. Thus overall these results indicate that RANKL induces osteoclastogenesis through the activation of NF-κB, and treatment with curcumin inhibits both the NF-κB activation and osteoclastogenesis induced by RANKL.

Induction of Cell Cycle Arrest and Apoptosis by the Proteasome Inhibitor PS-341 in Hodgkin Disease Cell Lines Is Independent of Inhibitor of Nuclear Factor-κB Mutations or Activation of the CD30, CD40, and RANK Receptors

Purpose: The malignant Hodgkin and Reed-Sternberg cells of Hodgkin disease (HD) are known to constitutively express high levels of activated nuclear factor κB (NF-κB), which plays an important role in their survival. The proteasome inhibitor PS-341 has been recently shown to modulate tumor cell proliferation and survival by inhibiting NF-κB and modulating critical cellular regulatory proteins, but its activity in cells carrying IκBα gene mutations has not been reported previously. Experimental Design: The activity of PS-341 in four well-characterized, HD-derived cell lines. Cell proliferation and apoptosis were determined by the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy-phenyl)-2-(4-sulfonyl)-2H-tetrazolium (MTS) and Annexin-V binding methods, respectively. Cell cycle analysis was determined by flow cytometry. Intracellular protein levels were determined by Western blot. Results: PS-341 demonstrated a strong antiproliferative activity, which was irrespective of the status of mutations in IκBα and even the presence of CD30, CD40, or RANK receptor activation. This effect was attributable to the induction of apoptosis and cell cycle arrest at the G2-M phase. PS-341 not only inhibited nuclear localization of NF-κB but also activated the caspase cascade, increased p21 and Bax levels, and decreased Bcl-2 levels. Furthermore, PS-341 enhanced the effect of gemcitabine chemotherapy and potentiated the effect of tumor necrosis factor-related apoptosis-inducing ligand/APO2L and two agonistic antibodies to tumor necrosis factor-related apoptosis-inducing ligand death receptors R1 and R2. Conclusions: The in vitro activity of PS-341 against HD-derived cell lines suggests that PS-341 may have a therapeutic value for the treatment of HD.

Evidence That Receptor Activator of Nuclear Factor (NF)-κB Ligand Can Suppress Cell Proliferation and Induce Apoptosis through Activation of a NF-κB-independent and TRAF6-dependent Mechanism

The receptor activator of NF-κB ligand (RANKL), a recently identified member of the tumor necrosis factor (TNF) superfamily, has been shown to induce osteoclastogenesis and dendritic cell survival. Most members of the TNF superfamily suppress cell proliferation and induce apoptosis, but whether RANKL does so is not known. We demonstrate that treatment of monocyte RAW 264.7 cells with RANKL induces dose-dependent growth inhibition (IC50 = 10 ng/ml) as determined by dye uptake and [3H]thymidine incorporation methods. Suppression of RANKL-induced NF-κB activation by dominant-negative IκBα or by the NEMO-peptide had no effect on RANKL-induced cell growth inhibition. Inhibition of RANKL-induced JNK activation, however, abolished the RANKL-induced apoptosis. Suppression of interaction of RANK with TRAF6 by TRAF6-binding peptide abrogated the anti-proliferative effects of RANKL, suggesting the critical role of TRAF6. Flow cytometric analysis of cells treated with RANKL showed accumulation of cells in G0/G1 phase of the cell cycle, and this accumulation correlated with a decline in the levels of cyclin D1, cyclin D3, and cyclin E and an increase in cyclin-dependent kinase inhibitor p27 (Kip). Flow cytometric analysis showed the presence of annexin V-positive cells in cultures treated with RANKL. RANKL-induced apoptosis was further confirmed using calcein AM/ethidium homodimer-1 dye and cleavage of poly(ADP-ribose) polymerase (PARP), procaspase 3, and procaspase 9; benzyloxycarbonyl-VAD, the pancaspase inhibitor, suppressed the PARP cleavage. Thus, overall, our studies indicate that RANKL can inhibit cell proliferation and induce apoptosis through a TRAF-6-dependent but NF-κB-independent mechanism.

Chemopreventive Agents Induce Suppression of Nuclear Factor-κB Leading to Chemosensitization

Abstract: Nuclear factor‐κB (NF‐κB), a transcription factor, is present normally in the cytoplasm as an inactive heterotrimer consisting of p50, p65, and IκBα subunits. When activated, NF‐κB translocates to the nucleus as a p50‐p65 heterodimer. This factor regulates the expression of various genes that control apoptosis, viral replication, tumorigenesis, various autoimmune diseases, and inflammation. NF‐κB has been linked to the development of carcinogenesis for several reasons. First, various carcinogens and tumor promoters have been shown to activate NF‐κB. Second, activation of NF‐κB has been shown to block apoptosis and promote proliferation. Third, the tumor microenvironment can induce NF‐κB activation. Fourth, constitutive expression of NF‐κB is frequently found in tumor cells. Fifth, NF‐κB activation induces resistance to chemotherapeutic agents. Sixth, several genes involved in tumor initiation, promotion, and metastasis are regulated by NF‐κB. Seventh, various chemopreventive agents have been found to down‐regulate the NF‐κB activation. All these observation suggest that NF‐κB could mediate tumorigenesis and thus can be used as a target for chemoprevention and for the treatment of cancer. Agents that suppress NF‐κB activation can suppress the expression of genes involved in carcinogenesis and tumorigenesis in vivo.

Inhibition of macrophage nitric oxide production by gangliosides derived from a spontaneous T cell lymphoma: the involved mechanisms

Gangliosides (DLG) derived from a spontaneous T cell lymphoma (Daltons lymphoma) have been shown to impair the ability of lipopolysaccharide-activated macrophages to produce nitric oxide (NO). However, the mechanism and nature of this effect is not known. In this investigation, we sought to (1) determine whether the inhibitory action of DLG on macrophages is through the modulation of inducible nitric oxide synthase (iNOS) expression and (2) identify the possible mechanisms and signal transduction events underlying the inhibitory action of DLG. Immunoblot analysis of DLG-treated macrophages showed a decrease in iNOS expression. DLG also inhibited the production of monokines interleukin-1 and tumor necrosis factor by macrophages. However, the DLG-induced inhibition was reversible in nature. Studies showed that DLG-induced inhibition of macrophage activation could be blocked by sodium orthovanadate, indicating a role of phosphatase activity in ganglioside-induced inhibition.

Functional Regulatory Role of STAT3 in HPV16-Mediated Cervical Carcinogenesis

Signal transducer and activator of transcription 3 (STAT3) is an oncogenic transcription factor constitutively active and aberrantly expressed in cervical cancer. However, the functional role of STAT3 in regulation of HPVs viral oncogene expression and downstream events associated with cervical carcinogenesis is not known. Our present study performed on HPV16-positive cervical cancer cell lines (SiHa and CaSki) and primary tumor tissues revealed a strong positive correlation of constitutively active STAT3 with expression of HPV16 E6 and E7 oncoproteins and a negative association with levels of p53 and pRB. Pharmacologic targeting of STAT3 expression in cervical cancer cell lines either by STAT3-specific siRNA or blocking its tyrosine phosphorylation by AG490 or curcumin led to dose-dependent accumulation of p53 and pRb in cervical cancer cells. Interestingly, the suppression of STAT3 expression or activation was associated with the gradual loss of HPV16 E6 and E7 expression and was accompanied by loss of cell viability. The viability loss was specifically high in HPV16-positive cells as compared to HPV negative C33a cells. These findings substantiate the regulatory role of STAT3 in HPV16-mediated cervical carcinogenesis. Leads obtained from the present study provide a strong rationale for developing novel STAT3-based approaches for therapeutic interventions against HPV infection to control cervical cancer.

Deregulation of microRNAs Let-7a and miR-21 mediate aberrant STAT3 signaling during human papillomavirus-induced cervical carcinogenesis: role of E6 oncoprotein

BackgroundAberrantly expressed and constitutively active STAT3 signaling plays a pivotal role in initiation and progression of human papillomavirus-induced cervical carcinogenesis. However, the underlying mechanism(s) responsible for pleiotropic effects of STAT3 signaling is poorly understood. In view of emerging regulatory role of microRNAs, Let-7a and miR-21 that may interact with STAT3 signaling and/or its downstream effectors, present study was designed in HPV16-positive cervical cancer cells to assess the functional contribution of these miRs in STAT3 signaling in cervical cancer.MethodsFunctional silencing of STAT3 signaling and HPV16 oncoprotein expression in SiHa cells was done by STAT3-specific and 16 E6 siRNAs. Pharmacological intervention of STAT3 was done using specific inhibitors like curcumin and stattic. Loss-of-function study of miR-21 using miR-21 inhibitor and gain-of-function study of let-7a was done using let-7a mimic in SiHa cells.ResultsFunctional silencing of STAT3 signaling in SiHa cells by STAT3-specific siRNA resulted in a dose-dependent decrease in cellular miR-21 level. Pharmacological intervention of STAT3 using specific inhibitors like curcumin and Stattic that abrogated STAT3 activation resulted in loss of cellular miR-21 pool. Contrary to this, specific targeting of miR-21 using miR-21 inhibitor resulted in an increased level of PTEN, a negative regulator of STAT3, and reduced active pSTAT3 level. Besides miR-21, restoration of cellular Let-7a using chemically synthesized Let-7a mimic reduced overall STAT3 level. Abrogation of HPV oncoprotein E6 by specific siRNA resulted in increased Let-7a but loss of miR-21 and a correspondingly reduced pSTAT3/STAT3 and elevated the level of cellular PTEN.ConclusionsOur results demonstrate existence of a functional loop involving Let-7a, STAT3 and miR-21 which were found potentially regulated by viral oncoprotein E6. Implications: miR-21 and Let-7a along with STAT3 may prove useful targets for pharmacological intervention for management of cervical cancer.