TM‐233, a novel analog of 1′‐acetoxychavicol acetate,induces cell death in myeloma cells by inhibiting both JAK/STAT and proteasome activities

Although the introduction of bortezomib and immunomodulatory drugs has led to improved outcomes in patients with multiple myeloma, the disease remains incurable. In an effort to identify more potent and well‐tolerated agents for myeloma, we have previously reported that 1′‐acetoxychavicol acetate (ACA), a natural condiment from South‐East Asia, induces apoptotic cell death of myeloma cells in vitro and in vivo through inhibition of NF‐κB‐related functions. Searching for more potent NF‐κB inhibitors, we developed several ACA analogs based on quantitative structure–activity relationship analysis. TM‐233, one of these ACA analogs, inhibited cellular proliferation and induced cell death in various myeloma cell lines with a lower IC₅₀ than ACA. Treatment with TM‐233 inhibited constitutive activation of JAK2 and STAT3, and then downregulated the expression of anti‐apoptotic Mcl‐1 protein, but not Bcl‐2 and Bcl‐xL proteins. In addition, TM‐233 rapidly decreased the nuclear expression of NF‐κB and also decreased the accumulation of cytosolic NF‐κB. We also examined the effects of TM‐233 on bortezomib‐resistant myeloma cells that we recently established, KMS‐11/BTZ and OPM‐2/BTZ. TM‐233, but not bortezomib, inhibited cellular proliferation and induced cell death in KMS‐11/BTZ and OPM‐2/BTZ cells. Interestingly, the combination of TM‐233 and bortezomib significantly induced cell death in these bortezomib‐resistant myeloma cells through inhibition of NF‐κB activity. These results indicate that TM‐233 could overcome bortezomib resistance in myeloma cells mediated through different mechanisms, possibly inhibiting the JAK/STAT pathway. In conclusion, TM‐233 might be a more potent NF‐κB inhibitor than ACA, and could overcome bortezomib resistance in myeloma cells.     

Anti‐proliferative and apoptosis‐inducing activity of lycopene against three subtypes of human breast cancer cell lines

Although lycopene, a major carotenoid component of tomatoes, has been suggested to attenuate the risk of breast cancer, the underlying preventive mechanism remains to be determined. Moreover, it is not known whether there are any differences in lycopene activity among different subtypes of human breast cancer cells. Using ER/PR positive MCF‐7, HER2‐positive SK‐BR‐3 and triple‐negative MDA‐MB‐468 cell lines, we investigated the cellular and molecular mechanism of the anticancer activity of lycopene. Lycopene treatment for 168 consecutive hours exhibited a time‐dependent and dose‐dependent anti‐proliferative activity against these cell lines by arresting the cell cycle at the G₀/G₁ phase at physiologically achievable concentrations found in human plasma. The greatest growth inhibition was observed in MDA‐MB‐468 where the sub‐G₀/G₁ apoptotic population was significantly increased, with demonstrable cleavage of PARP. Lycopene induced strong and sustained activation of the ERK1/2, with concomitant cyclin D1 suppression and p21 upregulation in these three cell lines. In triple negative cells, lycopene inhibited the phosphorylation of Akt and its downstream molecule mTOR, followed by subsequent upregulation of proapoptotic Bax without affecting anti‐apoptotic Bcl‐xL. Taken together, these data indicate that the predominant anticancer activity of lycopene in MDA‐MB‐468 cells suggests a potential role of lycopene for the prevention of triple negative breast cancer.     

Diosgenin targets Akt‐mediated prosurvival signaling in human breast cancer cells

In recent years, Akt signaling has gained recognition for its functional role in more aggressive, therapy‐resistant malignancies. As it is frequently constitutively active in cancer cells, several drugs are being investigated for their ability to inhibit Akt signaling. The purpose of this study is to determine effect of diosgenin (fenugreek), a dietary compound on Akt signaling and its downstream targets on estrogen receptor positive (ER⁺) and estrogen receptor negative (ER^?) breast cancer (BCa) cells. Diosgenin inhibits pAkt expression and Akt kinase activity without affecting PI3 kinase levels, resulting in the inhibition of its downstream targets, NF‐κB, Bcl‐2, survivin and XIAP. The Raf/MEK/ERK pathway, another functional downstream target of Akt, was inhibited by diosgenin in ER⁺ but not in ER^? BCa cells. Additionally, we found that diosgenin caused G1 cell cycle arrest by downregulating cyclin D1, cdk‐2 and cdk‐4 expression in both ER⁺ and ER^? BCa cells resulting in the inhibition of cell proliferation and induction of apoptosis. Interestingly, no significant toxicity was seen in the normal breast epithelial cells (MCF‐10A) following treatment with diosgenin. Additionally, in vivo tumor studies indicate diosgenin significantly inhibits tumor growth in both MCF‐7 and MDA‐231 xenografts in nude mice. Thus, these results suggest that diosgenin might prove to be a potential chemotherapeutic agent for the treatment of BCa. © 2009 UICC     

Enhancement of chemotherapeutic agent‐induced apoptosis by inhibition of NF‐κB using ursolic acid

NF‐κB activation is known to reduce the efficiency of chemotherapy in cancer treatment. Ursolic acid, a minimally toxic compound, has shown the capability to inhibit NF‐κB activation in living cells. Here, for the first time, we investigated the effects and mechanisms of NF‐κB inhibition by ursolic acid on chemotherapy treatment (Taxol or cisplatin) of cancer. ASTC‐a‐1 (human lung adenocarcinoma), Hela (human cervical cancer) cells, primary normal mouse cells of lung and liver and mouse in vivo model were used. Activity of signal factors (NF‐κB, Akt, Fas/FasL, BID, Bcl‐2, cytochrome c and caspase‐8, 3) was used to analyze the mechanisms of ursolic acid‐chemo treatment. Ursolic acid‐mediated suppression of NF‐κB drastically reduced the required dosage of the chemotherapeutic agents to achieve identical biological endpoints and enhanced the chemotherapeutic agent‐induced cancer cells apoptosis. Chemosensitization by ursolic acid in cancer cells was dependent on the amplified activation of intrinsic pathway (caspase‐8‐BID‐mitochondria‐cytochrome c‐caspase‐3) by augmentation of BID cleavage and activation of Fas/FasL‐caspase‐8 pathway. Prolonged treatment with relatively low doses of ursolic acid also sensitized cancer cells to the chemotherapeutic agents through suppression of NF‐κB. Chemosensitization by ursolic acid was observed only in cancer cells, but not in primary normal cells. The inhibitive effect of ursolic acid on NF‐κB was reversible, and the reversal was not accompanied by a loss in cells viability. By supplementing chemotherapy with minimally toxic ursolic acid, it is possible to improve the efficacy of cancer treatment by significantly reducing the necessary drug dose without sacrificing the treatment results.     

Chromosome instability in diffuse large B cell lymphomas is suppressed by activation of the noncanonical NF‐κB pathway

Diffuse large B cell lymphoma (DLBCL) is the most common form of lymphoma in the United States. DLBCL comprises biologically distinct subtypes including germinal center‐like (GCB) and activated‐B‐cell‐like DLBCL (ABC). The most aggressive type, ABC‐DLBCL, displays dysregulation of both canonical and noncanonical NF‐κB pathway as well as genomic instability. Although, much is known about the tumorigenic roles of the canonical NF‐kB pathway, the precise role of the noncanonical NF‐kB pathway remains unknown. Here we show that activation of the noncanonical NF‐κB pathway regulates chromosome stability, DNA damage response and centrosome duplication in DLBCL. Analysis of 92 DLBCL samples revealed that activation of the noncanonical NF‐κB pathway is associated with low levels of DNA damage and centrosome amplification. Inhibiting the noncanonical pathway in lymphoma cells uncovered baseline DNA damage and prevented doxorubicin‐induced DNA damage repair. In addition, it triggered centrosome amplification and chromosome instability, indicated by anaphase bridges, multipolar spindles and chromosome missegregation. We determined that the noncanonical NF‐κB pathway execute these functions through the regulation of GADD45α and REDD1 in a p53‐independent manner, while it collaborates with p53 to regulate cyclin G2 expression. Furthermore, this pathway regulates GADD45α, REDD1 and cyclin G2 through direct binding of NF‐κB sites to their promoter region. Overall, these results indicate that the noncanonical NF‐κB pathway plays a central role in maintaining genome integrity in DLBCL. Our data suggests that inhibition of the noncanonical NF‐kB pathway should be considered as an important component in DLBCL therapeutic approach.     

Receptor activator for nuclear factor‐κB ligand signaling promotes progesterone‐mediated estrogen‐induced mammary carcinogenesis

Breast cancer is a leading cause of cancer‐related death in women. Prolonged exposure to the ovarian hormones estrogen and progesterone increases the risk of breast cancer. Although estrogen is known as a primary factor in mammary carcinogenesis, very few studies have investigated the role of progesterone. Receptor activator for nuclear factor‐κB (NF‐κB) ligand (RANKL) plays an important role in progesterone‐induced mammary carcinogenesis. However, the molecular mechanism underlying RANKL‐induced mammary carcinogenesis remains unknown. In our current study, we show that RANKL induces glioma‐associated oncogene homolog 1 (GLI‐1) in estrogen‐induced progesterone‐mediated mammary carcinogenesis. In vivo experiments were carried out using ACI rats and in vitro experiments were carried out in MCF‐7 cells. In ACI rats, mifepristone significantly reduced the incidence of mammary tumors. Likewise, mifepristone also inhibited the proliferation of MCF‐7 cells. Hormone treatments induced RANKL, receptor activator of NF‐κB (RANK), and NF‐κB in a protein kinase B‐dependent manner and inhibited apoptosis by activation of anti‐apoptotic protein Bcl2 in mammary tumors and MCF‐7 cells. Mechanistic studies in MCF‐7 cells reveal that RANKL induced upstream stimulatory factor‐1 and NF‐κB, resulting in subsequent activation of their downstream target GLI‐1. We have identified that progesterone mediates estrogen‐induced mammary carcinogenesis through activation of GLI‐1 in a RANKL‐dependent manner.     

Platelet‐type 12‐lipoxygenase induces MMP9 expression and cellular invasion via activation of PI3K/Akt/NF‐κB

Prostate cancer is the most frequently diagnosed cancer and the second leading cause of death in males in the United States. Using human prostate cancer specimens, the authors have previously shown that elevated expression levels of 12‐lipoxygenase (12‐LOX) occurred more frequently in advanced stage, high‐grade prostate cancer, suggesting that 12‐LOX expression is associated with carcinoma progression and invasion. Previous reports from their group and others have shown that 12‐LOX is a positive modulator of invasion and metastasis; however, the mechanism remains unclear. In this work, a new link between 12‐LOX and the matrix metalloproteinase 9 (MMP9) in prostate cancer angiogenesis is reported. This study demonstrated that overexpression of 12‐LOX in prostate cancer PC‐3 cells resulted in elevated expression of MMP9 mRNA, protein and secretion. Exogenous addition of 12(S)‐hydroxy eicosatetraenoic acid, the sole and stable end product of arachidonic acid metabolism by 12‐LOX, is able to increase MMP9 expression in wild‐type PC‐3 cells. Furthermore, using pharmacological and genetic inhibition approaches, it was found that 12‐LOX activates phosphoinositol 3 kinase (PI3K)/Akt, which results in nuclear factor‐kappa B (NF‐κB)‐driven MMP9 expression, ensuing in enhanced chemoattraction of endothelial cells. Specific inhibitors of 12‐LOX, PI3K or NF‐κB inhibited MMP9 expression in 12‐LOX‐expressing PC‐3 cells and resulted in the blockade of the migratory ability of endothelial cells. In summary, the authors have identified a new pathway by which overexpression of 12‐LOX in prostate cancer cells leads to augmented production of MMP9 via activation of PI3K/Akt/NF‐κB signaling. The role of 12‐LOX‐mediated MMP9 secretion in endothelial cell migration may account for the proangiogenic function of 12‐LOX in prostate cancer.     

Curcumin inhibits the Sonic Hedgehog signaling pathway and triggers apoptosis in medulloblastoma cells

Medulloblastoma is an aggressive primary brain tumor that arises in the cerebellum of children and young adults. The Sonic Hedgehog (Shh) signaling pathway that plays important roles in the pathology of this aggressive disease is a promising therapeutic target. In the present report we have shown that curcumin has cytotoxic effects on medulloblastoma cells. Curcumin suppressed also cell proliferation and triggered cell‐cycle arrest at G₂/M phase. Moreover, curcumin inhibited the Shh–Gli1 signaling pathway by downregulating the Shh protein and its most important downstream targets GLI1 and PTCH1. Furthermore, curcumin reduced the levels of β‐catenin, the activate/phosphorylated form of Akt and NF‐κB, which led to downregulating the three common key effectors, namely C‐myc, N‐myc, and Cyclin D1. Consequently, apoptosis was triggered by curcumin through the mitochondrial pathway via downregulation of Bcl‐2, a downstream anti‐apoptotic effector of the Shh signaling. Importantly, the resistant cells that exhibited no decrease in the levels of Shh and Bcl‐2, were sensitized to curcumin by the addition of the Shh antogonist, cyclopamine. Furthermore, we have shown that curcumin enhances the killing efficiency of nontoxic doses of cisplatin and γ‐rays. In addition, we present clear evidence that piperine, an enhancer of curcumin bioavailability in humans, potentiates the apoptotic effect of curcumin against medulloblastoma cells. This effect was mediated through strong downregulation of Bcl‐2. These results indicate that curcumin, a natural nontoxic compound, represents great promise as Shh‐targeted therapy for medulloblastomas. © 2009 Wiley‐Liss, Inc.     

High IKKα expression is associated with reduced time to recurrence and cancer specific survival in oestrogen receptor (ER)‐positive breast cancer

The aim of our study was to examine the relationship between tumour IKKα expression and breast cancer recurrence and survival. Immunohistochemistry was employed in a discovery and a validation tissue microarray to assess the association of tumour IKKα expression and clinico‐pathological characteristics. After siRNA‐mediated silencing of IKKα, cell viability and apoptosis were assessed in MCF7 and MDA‐MB‐231 breast cancer cells. In both the discovery and validation cohorts, associations observed between IKKα and clinical outcome measures were potentiated in oestrogen receptor (ER) positive Luminal A tumours. In the discovery cohort, cytoplasmic IKKα was associated with disease‐free survival (p = 0.029) and recurrence‐free survival on tamoxifen (p < 0.001) in Luminal A tumours. Nuclear IKKα and a combination of cytoplasmic and nuclear IKKα (total tumour cell IKKα) were associated with cancer‐specific survival (p = 0.012 and p = 0.007, respectively) and recurrence‐free survival on tamoxifen (p = 0.013 and p < 0.001, respectively) in Luminal A tumours. In the validation cohort, cytoplasmic IKKα was associated with cancer‐specific survival (p = 0.023), disease‐free survival (p = 0.002) and recurrence‐free survival on tamoxifen (p = 0.009) in Luminal A tumours. Parallel experiment with breast cancer cells in vitro demonstrated the non‐canonical NF‐κB pathway was inducible by exposure to lymphotoxin in ER‐positive MCF7 cells and not in ER‐negative MDA‐MB‐231 cells. Reduction in IKKα expression by siRNA transfection increased levels of apoptosis and reduced cell viability in MCF7 but not in MDA‐MB‐231 cells. IKKα is an important determinant of poor outcome in patients with ER‐positive invasive ductal breast cancer and thus may represent a potential therapeutic target.     

Cancer stem cells in Epstein‐Barr virus‐associated gastric carcinoma

Cancer stem cells (CSCs) play a decisive role in the development and progression of cancer. To investigate CSCs in Epstein‐Barr virus (EBV)‐associated carcinoma (EBVaGC), we screened previously reported stem cell markers of gastric cancer in EBV‐infected gastric cancer cell lines (TMK1 and NUGC3) and identified CD44v6v9 double positive cells as candidate CSCs. CD44v6/v9^+/+ cells were sorted from EBVaGC cell line (SNU719) cells and EBV‐infected TMK1 cells and these cell populations showed high spheroid‐forming ability and tumor formation in SCID mice compared with the respective CD44v6/v9^?/? cells. Sphere‐forming ability was dependent on the nuclear factor‐κB (NF‐κB) signaling pathway, which was confirmed by decrease of sphere formation ability under BAY 11‐7082. Small interfering RNA knockdown of latent membrane protein 2A (LMP2A), one of the latent gene products of EBV infection, decreased spheroid formation in SNU719 cells. Transfection of the LMP2A gene increased the sphere‐forming ability of TMK1 cells, which was mediated through NF‐κB signaling. Together, these results indicate that CD44v6v9^+/+ cells are CSCs in EBVaGC that are maintained through the LMP2A/NF‐κB pathway. Future studies should investigate CD44v6/v9^+/+ cells in normal and neoplastic gastric epithelium to prevent and treat this specific subtype of gastric cancer infected with EBV.     

Betuletol 3‐methyl ether induces G2‐M phase arrest and activates the sphingomyelin and MAPK pathways in human leukemia cells

Betuletol 3‐methyl ether (BME) is a natural phenylbenzo‐γ‐pyrone that inhibits cell proliferation in human tumor cell lines and induces apoptotic cell death in HL‐60 cells. Here we show that BME displays strong cytotoxic properties in several human leukemia cell lines (U937, K‐562, THP‐1, Jurkat, and Molt‐3) and in cells that over‐express two anti‐apoptotic proteins, namely Bcl‐2 and Bcl‐x_L. BME arrested HL‐60 cells at G₂‐M phase of the cell cycle, which was associated with the accumulation of cyclin B1 and p21^Cip1. Fluorescence microscopy experiments suggest that BME blocked the cell cycle in mitosis. The in vivo tubulin polymerization assay shows that BME inhibits tubulin polymerization and causes similar changes of cellular microtubule network as colchicine. Our results demonstrate that BME‐induced cell death is (i) triggered in human myeloid leukemia cell that over‐express Bcl‐2 and Bcl‐x_L, and (ii) associated with loss of inner mitochondrial membrane potential (ΔΨm) and an increase in reactive oxygen species (ROS). Although ROS increased in response to BME, this did not seem to play a pivotal role in the apoptotic process since the anti‐oxidant trolox was unable to provide cell protection. The treatment of HL‐60 cells with BME induces the activation of mitogen‐activated protein kinases (MAPKs) such as c‐Jun N‐terminal kinases, p38 mitogen‐activated protein kinases and extracellular signal‐regulated kinases (ERK)1/2 and stimulates the acid sphingomyelinase with concomitant ceramide generation. The findings of this study suggest that BME could be useful in the development of novel anticancer agents. © 2009 Wiley‐Liss, Inc.     

DCLK1 promotes epithelial‐mesenchymal transition via the PI3K/Akt/NF‐κB pathway in colorectal cancer

Double cortin‐like kinase 1 (DCLK1) plays important roles during the epithelial‐mesenchymal transition (EMT) process in human colorectal cancer (CRC). However, the role of DCLK1 in regulating the EMT of CRC is still poorly understood. In this study, we report evidence that DCLK1 acts as a potent oncogene to drive its extremely malignant character of EMT in an NF‐κB‐dependent manner in CRC cells. Mechanistic investigations showed that DCLK1 induced the NF‐κBp65 subunit expression through the PI3K/Akt/Sp1 axis and activated NF‐κBp65 through the PI3K/Akt/IκBα pathway during the EMT of CRC cells. Moreover, we found that silencing the expression of DCLK1 inhibited the invasion and metastasis of CRC cells in vivo. Collectively, our findings identify DCLK1 as a pivotal regulator of an EMT axis in CRC, thus implicating DCLK1 as a potential therapeutic target for CRC metastasis.     

Growth‐stimulatory effect of resveratrol in human cancer cells

Earlier studies have shown that resveratrol could induce death in several human cancer cell lines in culture. Here we report our observation that resveratrol can also promote the growth of certain human cancer cells when they are grown either in culture or in athymic nude mice as xenografts. At relatively low concentrations (≤5 µM), resveratrol exerted a significant growth‐stimulatory effect in the MDA‐MB‐435s human cancer cells, but this effect was not observed in several other human cell lines tested. Analysis of cell signaling molecules showed that resveratrol induced the activation of JNK, p38, Akt, and NF‐κB signaling pathways in these cells. Further analysis using pharmacological inhibitors showed that only the NF‐κB inhibitor (BAY11‐7082) abrogated the growth‐stimulatory effect of resveratrol in cultured cells. In athymic nude mice, resveratrol at 16.5 mg/kg body weight enhanced the growth of MDA‐MB‐435s xenografts compared to the control group, while resveratrol at the 33 mg/kg body weight dose did not have a similar effect. Additional analyses confirmed that resveratrol stimulated cancer cell growth in vivo through activation of the NF‐κB signaling pathway. Taken together, these observations suggest that resveratrol at low concentrations could stimulate the growth of certain types of human cancer cells in vivo. This cell type‐specific mitogenic effect of resveratrol may also partly contribute to the procarcinogenic effect of alcohol consumption (rich in resveratrol) in the development of certain human cancers. © 2010 Wiley‐Liss, Inc.     

Toll‐like receptor‐9 agonists increase cyclin D1 expression partly through activation of activator protein‐1 in human oral squamous cell carcinoma cells

Increasing evidence suggests that malignant transformation can result from chronic infection, and Toll‐like receptors (TLRs) may play an important role in this process. We have previously reported that the increased expression of TLR‐9 is associated with tumor cell proliferation in oral cancer. However, the mechanisms involved have not been elucidated. The aim of this study was to investigate whether CpG‐oligodeoxynucleotides (CpG‐ODN), a special TLR‐9 agonist, is able to exert the proliferation‐promoting effect in human oral squamous cell carcinoma (OSCC), and to explore the possible underlying molecular mechanism. Flow cytometry, MTT, and colony formation assay were used to evaluate cell proliferation and cell cycle distribution. The mRNA and protein levels were analyzed by quantitative RT‐PCR and Western blot assay. Luciferase reporter gene, EMSA, and ChIP assays were used to detect the activity of activator protein‐1 (AP‐1) and nuclear factor‐κB (NF‐κB) in HB cells. Results showed that CpG‐ODN could stimulate proliferation of HB cells in a dose‐ and time‐dependent manner with a promoted G₁/S cell cycle progression. Increased cyclin D1 expression was detected in the nuclear region after CpG‐ODN treatment. Moreover, CpG‐ODN promoted nuclear translocation and activation of AP‐1, which appeared to be required for TLR‐9‐mediated cyclin D1 expression and subsequently cell proliferation, but seemed to have little impact on NF‐κB activity. Our results indicate that CpG‐ODN stimulates tumor cell proliferation through TLR‐9‐mediated AP‐1‐activated cyclin D1 expression in OSCC HB cells. Pharmacologic inhibition of the TLR‐9/AP‐1/cyclin D1 pathway may be a new therapeutic approach for prevention as well as treatment of OSCC.     

5,7,3′‐trihydroxy‐3,4′‐dimethoxyflavone‐induced cell death in human Leukemia cells is dependent on caspases and activates the MAPK pathway

Flavonoids are polyphenolic compounds which display a vast array of biological activities and are promising anticancer agents. In this study we investigated the effect of 5,7,3′‐trihydroxy‐3,4′‐dimethoxyflavone (THDF) on viability of nine human tumor cell lines and found that it was highly cytotoxic against leukemia cells. THDF induced G₂–M phase cell‐cycle arrest and apoptosis through a caspase‐dependent mechanism involving cytochrome c release, processing of multiple caspases (caspase‐3, ‐6, ‐7, and ‐9) and cleavage of poly(ADP‐ribose) polymerase. Overexpression of the protective mitochondrial proteins Bcl‐2 and Bcl‐x_L conferred partial resistance to THDF‐induced apoptosis. This flavonoid induced the phosphorylation of members of the mitogen‐activated protein kinases (MAPKs) family and cell death was attenuated by inhibition of c‐jun N‐terminal kinases/stress‐activated protein kinases (JNK/SAPK) and of extracellular signal‐regulated kinases (ERK) 1/2. In the present study we report that THDF‐induced cell death is mediated by an intrinsic dependent apoptotic event involving mitochondria and MAPKs, and through a mechanism independent of the generation of reactive oxygen species. The results suggest that THDF could be useful in the development of novel anticancer agents. Mol. Carcinog. © 2010 Wiley‐Liss, Inc.