CD44 promotes multi-drug resistance by protecting P-glycoprotein from FBXO21-mediated ubiquitination.

Here we demonstrate that a ubiquitin E3-ligase, FBXO21, targets the multidrug resistance transporter, ABCB1, also known as P-glycoprotein (P-gp), for proteasomal degradation. We also show that the Ser291-phosphorylated form of the multifunctional protein and stem cell marker, CD44, inhibits FBXO21-directed degradation of P-gp. Thus, CD44 increases P-gp mediated drug resistance and represents a potential therapeutic target in P-gp-positive cells.

Identification of function for CD44 intracytoplasmic domain (CD44-ICD): modulation of matrix metalloproteinase 9 (MMP-9) transcription via novel promoter response element.

CD44 is a multifunctional cell receptor that conveys a cancer phenotype, regulates macrophage inflammatory gene expression and vascular gene activation in proatherogenic environments, and is also a marker of many cancer stem cells. CD44 undergoes sequential proteolytic cleavages that produce an intracytoplasmic domain called CD44-ICD. However, the role of CD44-ICD in cell function is unknown. We take a major step toward the elucidation of the CD44-ICD function by using a CD44-ICD-specific antibody, a modification of a ChIP assay to detect small molecules, and extensive computational analysis. We show that CD44-ICD translocates into the nucleus, where it then binds to a novel DNA consensus sequence in the promoter region of the MMP-9 gene to regulate its expression. We also show that the expression of many other genes that contain this novel response element in their promoters is up- or down-regulated by CD44-ICD. Furthermore, hypoxia-inducible factor-1α (Hif1α)-responsive genes also have the CD44-ICD consensus sequence and respond to CD44-ICD induction under normoxic conditions and therefore independent of Hif1α expression. Additionally, CD44-ICD early responsive genes encode for critical enzymes in the glycolytic pathway, revealing how CD44 could be a gatekeeper of the Warburg effect (aerobic glycolysis) in cancer cells and possibly cancer stem cells. The link of CD44 to metabolism is novel and opens a new area of research not previously considered, particularly in the study of obesity and cancer. In summary, our results finally give a function to the CD44-ICD and will accelerate the study of the regulation of many CD44-dependent genes.

{beta}3-integrin expression on tumor cells inhibits tumor progression, reduces metastasis, and is associated with a favorable prognosis in patients with ovarian cancer.

The role of the vitronectin receptor (alpha(v)beta(3)-integrin) as a tumor promoter seems well established, and, consequently, therapies that block this integrin are currently in clinical testing. We undertook the current study to determine whether alpha(v)beta(3)-integrin is an appropriate target in ovarian cancer treatment. Expression of beta(3)-integrin in SKOV3ip1 ovarian cancer cells led to the overexpression of alpha(v)beta(3)-integrin on the cell surface and increased adhesion. However, alpha(v)beta(3)-integrin-overexpressing cells showed impaired invasion, protease expression, and colony formation. These results were recapitulated in xenograft studies: alpha(v)beta(3)-integrin-expressing cells showed increased adhesion to mouse peritoneum, but the overall number of metastatic nodules (105 versus 68 tumors) and tumor weight were significantly lower than those in the parental SKOV3ip1 cells. The alpha(v)beta(3)-integrin-overexpressing cells had a decreased proliferation rate mediated by inhibition of cyclin B1 and induction of phospho-Cdc2 and p53 expression, consistent with a G(2)M cell cycle arrest. Confirming the above results, inhibition of beta(3)-integrin in cultured or primary OvCa cells decreased adhesion but increased invasion and proliferation. Patients with tumors expressing high beta(3)-integrin had significantly better disease-free and overall survival (52 months versus 27 months, P < 0.05). This study shows that alpha(v)beta(3)-integrin expression on tumor cells actually slows tumor progression and acts as a tumor suppressor. Therefore, the vitronectin receptor might not be an appropriate therapeutic target in ovarian cancer.

Nuclear-factor-kappaB (NF-kappaB) and radical oxygen species play contrary roles in transforming growth factor-beta1 (TGF-beta1)-induced apoptosis in hepatocellular carcinoma (HCC) cells.

Nuclear-Factor-kappaB (NF-kappaBeta can counteract transforming growth factor-beta1 (TGF-beta1)-induced apoptosis in malignant hepatocytes through up-regulation of its downstream genes, such as X-linked inhibitor of apoptosis protein (XIAP). Reports have demonstrated that TGF-beta1 can induce oxidative stress, and c-Jun N-terminal Kinase1 (JNK1) is indispensable for TGF-beta1-induced apoptosis pathway, but the relationship between radical oxygen species (ROS) and the activation of JNKs is still unclear. In the present study, we found that ROS can induce JNK activation in TGF-beta1 mediated apoptosis in hepatocytes. The inhibitors of hydrogen peroxide and superoxide, which were produced by mitochondria under stress, could inhibit the phosphorylation of c-Jun in XIAP knockdown cells. In conclusion, it is the first time to show that both NF-kappaB and antioxidants can counteract TGF-beta1-induced apoptosis in hepatic cell death through JNK1 pathway.

The initial steps of ovarian cancer cell metastasis are mediated by MMP-2 cleavage of vitronectin and fibronectin.

Most patients (80%) with ovarian cancer (OvCa) present with metastatic disease. Attachment of OvCa cells to peritoneum and omentum represents the first rate-limiting step for metastatic spread. Therefore, identifying factors regulating cell attachment in the abdominal cavity is critical to the development of therapeutic agents. We show here that MMP-2 expression was upregulated in OvCa cells upon attachment to their microenvironment. Downregulation of MMP-2 mRNA or pharmacological inhibition of MMP-2 proteolytic function, in both human OvCa primary cells and cell lines, reduced attachment of OvCa cells to a 3D organotypic model of metastatic OvCa, full human omentum or peritoneum, and in vivo to mouse peritoneum and omentum. Absence of MMP-2 in the host did not alter OvCa adhesion, as determined utilizing mice harboring homozygous null mutations in either the Mmp2 or Mmp9 genes. Conversely, adhesion induced upregulation of MMP-2 mRNA in OvCa cells. MMP-2 inhibition in OvCa cells through pharmacological or antibody treatment prior to i.p. dissemination in nude mice significantly decreased tumor growth and metastasis and extended survival. MMP-2 enhanced peritoneal adhesion of OvCa cells through cleavage of ECM proteins fibronectin (FN) and vitronectin (Vn) into small fragments and increased binding of OvCa cells to these FN and Vn fragments and their receptors, alpha5beta1 and alphaVbeta3 integrin. These findings indicate that MMP-2 expressed by metastatic OvCa cells functionally regulates their attachment to peritoneal surfaces.

X-linked inhibitor of apoptosis (XIAP) inhibits c-Jun N-terminal kinase 1 (JNK1) activation by transforming growth factor beta1 (TGF-beta1) through ubiquitin-mediated proteosomal degradation of the TGF-beta1-activated kinase 1 (TAK1).

Active NF-kappaB renders malignant hepatocytes refractory to the growth inhibitory and pro-apoptotic properties of transforming growth factorbeta1 (TGF-beta1). NF-kappaB counteracts TGF-beta1-induced apoptosis through up-regulation of downstream target genes, such as XIAP and Bcl-X(L), which in turn inhibit the intrinsic pathway of apoptosis. In addition, induction of NF-kappaB by TGF-beta1 inhibits JNK signaling, thereby attenuating TGF-beta1-induced cell death of normal hepatocytes. However, the mechanism involved in the negative cross-talk between the NF-kappaB and JNK pathways during TGF-beta1 signaling has not been determined. In this study, we have identified the XIAP gene as one of the critical mediators of NF-kappaB-mediated suppression of JNK signaling. We show that NF-kappaB plays a role in the up-regulation of XIAP gene expression in response to TGF-beta1 treatment and forms a TGF-beta1-inducible complex with TAK1. Furthermore, we show that the RING domain of XIAP mediates TAK1 polyubiquitination, which then targets this molecule for proteosomal degradation. Down-regulation of TAK1 protein expression inhibits TGF-beta1-mediated activation of JNK and apoptosis. Conversely, silencing of XIAP promotes persistent JNK activation and potentiates TGF-beta1-induced apoptosis. Collectively, our findings identify a novel mechanism for the regulation of JNK activity by NF-kappaB during TGF-beta1 signaling and raise the possibility that pharmacologic inhibition of the NF-kappaB/XIAP signaling pathway might selectively abolish the pro-oncogenic activity of TGF-beta1 in advanced hepatocellular carcinomas (HCCs) without affecting the pro-apoptotic effects of TGF-beta1 involved in normal liver homeostasis.

Evaluation of genotoxicity of medicinal plant extracts by the comet and VITOTOX tests.

We report the results of our genotoxic evaluation of extracts from three medicinal plants Acacia nilotica, Juglans regia, and Terminalia chebula and the herbal drug Triphala employing the VITOTOX and comet tests.These tests detect DNA damage in prokaryotic and eukaryotic test systems, respectively. In the VITOTOX test, none of the extracts were identified as genotoxic. In the comet assay, extracts of Acacia nilotica showed statistically significant DNA damage only in a concentration of 2500 ppm (highest tested dose), whereas extracts from Juglans regia showed significant damage in concentrations above 250 ppm and more. Extracts from Terminalia chebula and Tripahala significantly increased DNA damage in a concentration above 500 ppm. This is not considered contradictory, because DNA damage in the alkaline comet assay may not be permanent and hence may not necessarily result in mutations. All the extracts were previously found in the Ames assay to have potent antimutagenic effects against the direct acting mutagens NPD, sodium azide, and the S9-dependent mutagen 2-AF. The results of the previous study using the Ames assay are in conformity with those of the VITOTOX test. It was found that the extracts were safe in concentrations of up to 1000 microg/0.1 mL and 2500 microg/0.1 mL. A literature survey also showed that plant extracts can be mutagenic as well as antimutagenic depending on the test system used. This indicates that a battery of assays is needed before any conclusion can be reached.

Transforming growth factor-alpha inhibits the intrinsic pathway of c-Myc-induced apoptosis through activation of nuclear factor-kappaB in murine hepatocellular carcinomas.

Nuclear factor-kappaB (NF-kappaB) plays an important role during liver neoplastic development through transcriptional regulation of prosurvival genes, which then counteract the death-inducing signals elicited by the host immune response. The c-Myc proto-oncogene is frequently deregulated in liver tumors. Furthermore, enforced expression of c-Myc in the liver promotes the development of hepatocellular carcinomas, a process that is accelerated by coexpression with transforming growth factor-alpha (TGF-alpha). TGF-alpha/c-Myc-derived hepatocellular carcinomas display reduced apoptotic levels compared with those of single c-Myc transgenic hepatocellular carcinomas, suggesting that TGF-alpha provides a survival advantage to c-Myc-transformed hepatocytes. Given that TGF-alpha/c-Myc hepatocellular carcinomas display constitutive NF-kappaB activity, here, we have tested the hypothesis that enforced expression of TGF-alpha results in constitutive NF-kappaB activation and enhanced cell survival using TGF-alpha/c-Myc-derived hepatocellular carcinoma cell lines. We show that TGF-alpha induces NF-kappaB through the phosphatidylinositol 3-kinase/Akt axis in these bitransgenic hepatocellular carcinomas. Furthermore, we found that adenovirus-mediated inhibition of NF-kappaB activity impairs the ability of TGF-alpha/c-Myc-derived tumor cells to grow in an anchorage-independent fashion due to sensitization to c-Myc-induced apoptosis. Lastly, we show that NF-kappaB inhibits c-Myc-induced activation of caspase-9 and caspase-3 through up-regulation of the antiapoptotic target genes Bcl-X(L) and X-linked inhibitor of apoptosis (XIAP). Overall, these results underscore a crucial role of NF-kappaB in disabling apoptotic pathways initiated by oncogenic transformation.

The in vitro cytotoxic and apoptotic activity of Triphala--an Indian herbal drug.

A study on cytotoxic effect of acetone extract of "Triphala" whose antimutagenicity has already been tested. The in vitro antimutagenic activity of Triphala--an Indian herbal drug. Food Chemistry and Toxicology 40, 47-54) was extended to test its cytotoxic effects on cancer cell-lines using Shionogi 115 (S115) and MCF-7 breast cancer cells and PC-3 and DU-145 prostate cancer cells as models. The results revealed that acetone extract of "Triphala" showed a significant cytotoxic effect on these cancer cell-lines and the effect was similar on all cancer cell lines used in this study. The major phenolic compounds in the most potent acetone extracts were isolated and purified. Structural analysis was conducted using spectroscopic techniques including mass spectroscopy, nuclear magnetic resonance (NMR) and infrared (IR) which showed gallic acid as the major component. The suppression of the growth of cancer cells in cytotoxic assays may be due to the gallic acid-a major polyphenol observed in "Triphala".

Regulation of alpha-fetoprotein by nuclear factor-kappaB protects hepatocytes from tumor necrosis factor-alpha cytotoxicity during fetal liver development and hepatic oncogenesis.

Nuclear factor-kappaB (NF-kappaB) plays a critical role during fetal liver development and hepatic oncogenesis. Here, we have assessed whether NF-kappaB activity is required for murine hepatocellular carcinoma cell survival. We show that adenoviral-mediated inhibition of inhibitor of NF-kappaB kinase-beta (IKK-2) activity in hepatocellular carcinomas derived from transforming growth factor (TGF)-alpha/c-myc bitransgenic mice leads to inhibition of NF-kappaB and promotes tumor necrosis factor (TNF)-alpha-mediated cell death of malignant hepatocytes but not the surrounding peritumorous tissue. Induction of apoptosis is accompanied by inhibition of Bcl-X(L) and XIAP, two pro-survival NF-kappaB target genes. In addition, we have identified the alpha-fetoprotein (AFP) as a novel downstream target of NF-kappaB. We show that repression of IKK-2 activity in hepatocellular carcinomas promotes down-regulation of AFP gene expression. Likewise, genetic disruption of the RelA subunit results in reduced AFP gene expression during embryonic liver development, at a time in which fetal hepatocytes are sensitized to TNF-alpha-mediated cell killing. In this regard, we show that AFP inhibits TNF-alpha-induced cell death of murine hepatocellular carcinomas through association with TNF-alpha and inhibition of TNFRI signaling. Thus, NF-kappaB-mediated regulation of AFP gene expression during liver tumor formation and embryonic development of the liver constitutes a potential novel mechanism used by malignant and fetal hepatocytes to evade immune surveillance.

ATM and the catalytic subunit of DNA-dependent protein kinase activate NF-kappaB through a common MEK/extracellular signal-regulated kinase/p90(rsk) signaling pathway in response to distinct forms of DNA damage.

We have identified a novel pathway of ataxia telangiectasia mutated (ATM) and DNA-dependent protein kinase (DNA-PK) signaling that results in nuclear factor kappaB (NF-kappaB) activation and chemoresistance in response to DNA damage. We show that the anthracycline doxorubicin (DOX) and its congener N-benzyladriamycin (AD 288) selectively activate ATM and DNA-PK, respectively. Both ATM and DNA-PK promote sequential activation of the mitogen-activated protein kinase (MAPK)/p90(rsk) signaling cascade in a p53-independent fashion. In turn, p90(rsk) interacts with the IkappaB kinase 2 (IKK-2) catalytic subunit of IKK, thereby inducing NF-kappaB activity and cell survival. Collectively, our findings suggest that distinct members of the phosphatidylinositol kinase family activate a common prosurvival MAPK/IKK/NF-kappaB pathway that opposes the apoptotic response following DNA damage.

Bioassay-guided isolation of antimutagenic factors from fruits of Terminalia bellerica.

In the course of our search for novel polyphenolic antimutagenic agents from medicinal plants, we examined water, acetone, and chloroform extracts of Terminalia bellerica for their antimutagenic potency using the Ames Salmonella/microsome assay. Acetone extract exhibited variable inhibitory activity of 65.6%, and 69.7% with 4-O-nitrophenylenediamine (NPD) and sodium azide, respectively (as direct-acting mutagens), and 81.4% with 2-aminofluorene (2AF) (an S9-dependent mutagen), in the preincubation mode of experimentation. Inhibition with chloroform and water extracts was rather insignificant. Studies are well underway to isolate and identify the active polyphenolic compounds from acetone extract, which could be used as effective chemopreventive agents in the future.

Indian medicinal plants as a reservoir of protective phytochemicals.

India is one of the 12 mega diversity countries in the world so it has a vital stake in conservation and sustainable utilization of its biodiversity resources. Plant secondary metabolites have been of interest to man for a long time due to their pharmacological relevance. With this in view, the bark powder of Acacia auriculiformis, A. nilotica, Juglans regia, and the fruit powder of Terminalia bellerica, T. chebula, Emblica officinalis, and a combination drug "Triphala," which are known to be rich in polyphenols, were tested for their antimutagenic activities. Antimutagenic activities of the extracts were estimated by employing the plate incorporation Ames Salmonella histidine reversion assay by using the frame shift mutagen tester strain TA98 and base pair substitution strain TA100 against direct acting mutagens (NPD, sodium azide), and the S9-dependent mutagen 2-aminofluorene(2AF). Acetone extracts of all the plants exhibited significant antimutagenic activities among the other extracts tested, but an acetone extract of Acacia nilotica showed a marked anti-mutagent effect. Furthermore, it was more effective against indirect acting mutagen, 2AF, in both TA98 and TA100 tester strains of Salmonella typhimurium than against the direct acting mutagens. The results indicate that an acetone extract of bark and fruit of the medicinal plants under study harbors constituents with promising antimutagenic/anticarcinogenic potential that could be investigated further.

A correlative study on antimutagenic and chemopreventive activity of Acacia auriculiformis A. Cunn. and Acacia nilotica (L.) Willd. Ex Del.

The present study provides a correlation of the antimutagenic and chemopreventive activity of the barks of two commonly observed plants viz. Acacia auriculiformis and Acacia nilotica. We used the Ames antimutagenicity assay and the mouse mammary gland organ culture (MMOC) model. The plants were extracted with organic solvents to obtain chloroform fractions and acetone extracts. The antimutagenic activity was determined in two different strains using both direct-acting [4-nitro-o-phenylenediamine (NPD) or sodium azide] and indirect-acting [2-aminofluorene (2AF)] mutagens. The anticarcinogenic activity was evaluated based on the development of preneoplastic lesions in response to the chemical carcinogen 7,12-dimethylbenz[a]anthracene (DMBA). The results showed that the activity resulting from the 2AF mutagen was selectively greater than the activity from the direct-acting mutagens. Moreover, in general, acetone extracts were more potent in suppressing mutagenesis than the chloroform extracts. The antimutagenicity results obtained with extracts using the 2AF--TA100 system were comparable to the chemopreventive results with DMBA-induced mammary lesions. The order of activity in both tests was A. nilotica > A. auriculiformis. These results exhibited a good correlation between the antimutagenesis assay and the MMOC model, suggesting that these plants may contain active chemopreventive agents.