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Human epidermal growth factor receptor 2 (HER2)-positive breast cancer exhibits early relapses, poor prognoses, and high recurrence rates. Herein, a JNK-targeting compound has been developed that may be of utility in HER2-positive mammary carcinoma. The design of a pyrimidine-and coumarin-linked structure targeting JNK was explored and the lead structure PC-12 [4-(3-((2-((4-chlorobenzyl)thio) pyrimidin-4-yl)oxy)propoxy)-6-fluoro-2 H -chromen-2-one ( 5d )] was observed to selectively inhibit the proliferation of HER2-positive BC cells. The compound PC-12 exerted DNA damage and induced apoptosis in HER-2 positive BC cells more significantly compared to HER-2 negative BC cells. PC-12 induced PARP cleavage and down-regulated the expression of IAP-1, BCL-2, SURVIVIN, and CYCLIN D1 in BC cells. In silico and theoretical calculations showed that PC-12 could interact with JNK, and in vitro studies demonstrated that it enhanced JNK phosphorylation through ROS generation. Overall, these findings will assist the discovery of new compounds targeting JNK for use in HER2-positive BC cells.

Daidzin (DDZ) extracted from Pueraria lobate (Fabaceae) is a widely known phytoestrogen. DDZ can display anti-cancer activities against breast and prostate cancers, but its anti-oncogenic actions in multiple myeloma (MM) cells have not been studied. The signal transducer and activator of transcription 3 (STAT3) can control key processes including proliferation, differentiation, and survival in MM cells. Here, we noted that DDZ abrogated STAT3 activation (both constitutive as well as inducible) at Tyr705 and Ser727 in MM cells. Additionally, DDZ mitigated the phosphorylation of STAT3 upstream Janus-activated kinases (JAK1/2) and c-Src kinases. Pervanadate (tyrosine phosphatase blocker) exposure altered the DDZ-induced inhibition of STAT3 activation, thus affecting the action of this phytoestrogen on apoptosis. Moreover, DDZ impeded proliferation and augmented the apoptotic effects of bortezomib (Bor) in MM cells. Overall, the data indicate that DDZ may act as a potent suppressor of STAT3 signaling cascade, and the co-treatment of DDZ and Bor could be a promising therapeutic strategy, specifically in MM.

Reactive oxygen species (ROS) play a pivotal role in biological processes and continuous ROS production in normal cells is controlled by the appropriate regulation between the silver lining of low and high ROS concentration mediated effects. Interestingly, ROS also dynamically influences the tumor microenvironment and is known to initiate cancer angiogenesis, metastasis, and survival at different concentrations. At moderate concentration, ROS activates the cancer cell survival signaling cascade involving mitogen-activated protein kinase/extracellular signal-regulated protein kinases 1/2 (MAPK/ERK1/2), p38, c-Jun N-terminal kinase (JNK), and phosphoinositide-3-kinase/ protein kinase B (PI3K/Akt), which in turn activate the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), matrix metalloproteinases (MMPs), and vascular endothelial growth factor (VEGF). At high concentrations, ROS can cause cancer cell apoptosis. Hence, it critically depends upon the ROS levels, to either augment tumorigenesis or lead to apoptosis. The major issue is targeting the dual actions of ROS effectively with respect to the concentration bias, which needs to be monitored carefully to impede tumor angiogenesis and metastasis for ROS to serve as potential therapeutic targets exogenously/endogenously. Overall, additional research is required to comprehend the potential of ROS as an effective anti-tumor modality and therapeutic target for treating malignancies.

Casticin (CTC), one of the major components of Vitex rotundifolia L., has been reported to exert significant beneficial pharmacological activities and can function as an antiprolactin, anticancer, anti-inflammatory, neuroprotective, analgesic, and immunomodulatory agent. This study aimed at investigating whether the proapoptotic effects of CTC may be mediated through the abrogation of signal transducers and activators of transcription-3 (STAT3) signaling pathway in a variety of human tumor cells. We found that CTC significantly decreased cell viability in a concentration-dependent manner and suppressed cell proliferation in 786-O, YD-8, and HN-9 cells. CTC also induced programmed cell death that was found to be mediated via caspase-3 activation and induction of poly(ADP-ribose) polymerase cleavage. Interestingly, CTC repressed both constitutive and interleukin-6-induced STAT3 activation in 786-O and YD-8 cells but only affected constitutive STAT3 phosphorylation in HN-9 cells. Moreover, CTC could potentiate ionizing radiation-induced apoptotic effects leading to the downregulation of STAT3 activation and thus may be used in combination with radiation against diverse malignancies., (© 2018 Wiley Periodicals, Inc.)

Background: Cycloastragenol (CAG), a triterpene aglycone is commonly prescribed for treating hypertension, cardiovascular disease, diabetic nephropathy, viral hepatitis, and various inflammatory-linked diseases., Hypothesis: We investigated CAG for its action on signal transducer and activator of transcription 3 (STAT3) activation cascades, and its potential to sensitize gastric cancer cells to paclitaxel-induced apoptosis., Methods: The effect of CAG on STAT3 phosphorylation and other hallmarks of cancer was deciphered using diverse assays in both SNU-1 and SNU-16 cells., Results: We observed that CAG exhibited cytotoxic activity against SNU-1 and SNU-16 cells to a greater extent as compared to normal GES-1 cells. CAG predominantly caused negative regulation of STAT3 phosphorylation at tyrosine 705 through the abrogation of Src and Janus-activated kinases (JAK1/2) activation. We noted that CAG impaired translocation of STAT3 protein as well as its DNA binding activity. It further decreased cellular proliferation and mediated its anticancer effects predominantly by causing substantial apoptosis rather than autophagy. In addition, CAG potentiated paclitaxel-induced anti-oncogenic effects in gastric tumor cells., Conclusions: Our results indicate that CAG can function to impede STAT3 activation in human gastric tumor cells and therefore it may be a suitable candidate agent for therapy of gastric cancer., (Copyright © 2019. Published by Elsevier GmbH.)

Chemoresistance is a leading obstacle in effective management of advanced prostate cancer (PCa). A better understanding of the molecular mechanisms involved in PCa chemoresistance could improve treatment of patients with PCa. In the present study, using immune histochemical, chemistry, and precipitation assays with cells from individuals with benign or malignant prostate cancer or established PCa cell lines, we found that the oncogenic transcription factor pre-B cell leukemia homeobox-1 (PBX1) promotes PCa cell proliferation and confers to resistance against common anti-cancer drugs such as doxorubicin and cisplatin. We observed that genetic PBX1 knockdown abrogates this resistance, and further experiments revealed that PBX1 stability was modulated by the ubiquitin-proteasomal pathway. To directly probe the impact of this pathway on PBX1 activity, we screened for PBX1-specific deubiquitinases (Dubs) and found that ubiquitin-specific peptidase 9 X-linked (USP9x) interacted with and stabilized the PBX1 protein by attenuating its Lys-48-linked polyubiquitination. Moreover, the USP9x inhibitor WP1130 markedly induced PBX1 degradation and promoted PCa cell apoptosis. The results in this study indicate that PBX1 confers to PCa chemoresistance and identify USP9x as a Dub of PBX1. We concluded that targeting the USP9x/PBX1 axis could be a potential therapeutic strategy for managing advanced prostate cancer., (© 2019 Liu et al.)

Oleuropein is one of the most abundant phenolic compounds found in olives. Epidemiological studies have indicated that an increasing intake of olive oil can significantly reduce the risk of breast cancer. However, the potential effect(s) of oleuropein on estrogen receptor (ER)-negative breast cancer is not fully understood. This study aims to understand the anticancer effects and underlying mechanism(s) of oleuropein on ER-negative breast cancer cells in vitro. The effect of oleuropein on the viability of breast cancer cell lines was examined by mitochondrial dye-uptake assay, apoptosis by flow cytometric analysis, nuclear factor-κB (NF-κB) activation by DNA binding/reporter assays and protein expression by Western blot analysis. In the present report, thiazolyl blue tetrazolium bromide assay results indicated that oleuropein inhibited the viability of breast cancer cells, and its effects were more pronounced on MDA-MB-231 as compared with MCF-7 cells. It was further found that oleuropein increased the level of reactive oxygen species and also significantly inhibited cellular migration and invasion. In addition, the activation of NF-κB was abrogated as demonstrated by Western blot analysis, NF-κB-DNA binding, and luciferase assays. Overall, the data indicates that oleuropein can induce substantial apoptosis via modulating NF-κB activation cascade in breast cancer cells., (© 2018 Wiley Periodicals, Inc.)

Background: Arctiin is a main component from the fruits of Arctium lappa L., that can be prescribed for cold or flu in East Asian countries; it has also been found to exert chemopreventive actions against various tumor cells., Hypothesis: In view of this evidence, we examined arctiin for its ability to trigger apoptosis and inhibit the activation of signal transducer and activator of transcription 3 (STAT3) in human multiple myeloma (MM) cells., Methods: We evaluated the effect of arctiin on STAT3 signaling cascades and its regulated functional responses in MM cells., Results: Arctiin effectively blocked the constitutive activation of STAT3 phosphorylation in the residue of tyrosine 705. Arctiin also abrogated the constitutive activation of Src phosphorylation and Janus-activated kinases (JAKs) 1/2. Furthermore, it was found that arctiin treatment clearly enhanced the mRNA and protein levels of protein tyrosine phosphatase ε (PTPε), and the silencing of PTPε caused a reversal of the arctiin-induced PTPε expression and the blockadge of STAT3 phosphorylation. Interestingly, arctiin could not repress IL-6-induced STAT3 activation in serum-starved U266 cells and when arctiin was incubated with a complete culture medium in RPMI 8226 and MM.1S cells. Arctiin suppressed cell proliferation, accumulated cells in the G2/M cell-cycle phase, and induced apoptosis within U266 cells, although the knockdown of PTPε prevented PARP cleavage and caspase-3 activation induced by the arctiin. In addition, arctiin exerted cytotoxicity in MM cells, but did not do so in peripheral blood mononuclear cells. Arctiin down-modulated diverse oncogenic gene products regulated by STAT3, although the induction of apoptosis by arctiin was abrogated upon transfection with pMXs-STAT3C in mouse embryonic fibroblast (MEF) cells. Arctiin also potentiated bortezomib-induced antitumor effects in U266 cells., Conclusion: On the whole, our results indicate that arctiin is a potentially new inhibitor of constitutive STAT3 activation through the induction of PTPε in MM, cells and therefore has great value in treating various tumors sheltering constitutively activated STAT3., (Copyright © 2018 Elsevier GmbH. All rights reserved.)

Novel and alternative options are being adopted to combat the initiation and progression of human cancers. One of the approaches is the use of molecules isolated from traditional medicinal herbs, edible dietary plants and seeds that play a pivotal role in the prevention/treatment of cancer, either alone or in combination with existing chemotherapeutic agents. Compounds that modulate these oncogenic processes are potential candidates for cancer therapy and may eventually make it to clinical applications. Diosgenin is a naturally occurring steroidal sapogenin and is one of the major bioactive compounds found in dietary fenugreek ( Trigonella foenum-graecum ) seeds. In addition to being a lactation aid, diosgenin has been shown to be hypocholesterolemic, gastro- and hepato-protective, anti-oxidant, anti-inflammatory, anti-diabetic, and anti-cancer. Diosgenin has a unique structural similarity to estrogen. Several preclinical studies have reported on the pro-apoptotic and anti-cancer properties of diosgenin against a variety of cancers, both in in vitro and in vivo. Diosgenin has also been reported to reverse multi-drug resistance in cancer cells and sensitize cancer cells to standard chemotherapy. Remarkably, diosgenin has also been reported to be used by pharmaceutical companies to synthesize steroidal drugs. Several novel diosgenin analogs and nano-formulations have been synthesized with improved anti-cancer efficacy and pharmacokinetic profile. In this review we discuss in detail the multifaceted anti-cancer properties of diosgenin that have found application in pharmaceutical, functional food, and cosmetic industries; and the various intracellular molecular targets modulated by diosgenin that abrogate the oncogenic process.

2,5-Dihydroxyacetophenone (DHAP) is an active compound obtained from Radix rehmanniae preparata, which is widely used as a herbal medicine in many Asian countries. DHAP has been found to possess anti-inflammatory, anti-anxiety, and neuroprotective qualities. For the present study, we evaluated the anti-cancer effects of DHAP on multiple myeloma cells. It was discovered that DHAP downregulated the expression of oncogenic gene products like Bcl-xl, Bcl-2, Mcl-1, Survivin, Cyclin D1, IAP-1, Cyclin E, COX-2, and MMP-9, and upregulated the expression of Bax and p21 proteins, consistent with the induction of G2/M phase cell cycle arrest and apoptosis in U266 cells. DHAP inhibited cell proliferation and induced apoptosis, as characterized by the cleavage of PARP and the activation of caspase-3, caspase-8, and caspase-9. Mitogen-activated protein kinase (MAPK) pathways have been linked to the modulation of the angiogenesis, proliferation, metastasis, and invasion of tumors. We therefore attempted to determine the effect of DHAP on MAPK signaling pathways, and discovered that DHAP treatment induced a sustained activation of JNK, ERK1/2, and p38 MAPKs. DHAP also potentiated the pro-apoptotic and anti-proliferative effects of bortezomib in U266 cells. Our results suggest that DHAP can be an effective therapeutic agent to target multiple myeloma., Competing Interests: The authors declare no conflicts of interest.

Isorhynchophylline (Rhy) is an active pharmacological component of Uncaria rhynchophylla that has been reported previously to exert significant antihypertensive and neuroprotective effects. However, very little is known about its potential anti-cancer activities. This study was carried out to evaluate the anticancer effects of Rhy against various human carcinoma cell lines. We found that Rhy exhibited substantial cytotoxic effect against human hepatocellular carcinoma HepG2 cells when compared with other human carcinoma cell lines including those of lung, pancreas, prostate, head and neck, breast, multiple myeloma, brain and renal cell carcinoma. Rhy induced apoptosis as characterized by accumulation of cells in sub G1 phase; positive Annexin V binding; activation of caspase-8, -9, and -3; and cleavage of PARP (poly-ADP ribose polymerase). This effect of Rhy correlated with the down-regulation of various proteins that mediated cell proliferation, cell survival, metastasis, and angiogenesis. Moreover, cell proliferation, migration, and constitutive CXCR4 (C-X-C chemokine receptor type 4), MMP-9 (Matrix metallopeptidase-9), and MMP-2 expression were inhibited upon Rhy treatment. We further investigated the effect of Rhy on the oncogenic cell signaling cascades through phospho-kinase array profiling assay. Rhy was found to abrogate phospho-p38, ERK, JNK, CREB, c-Jun, Akt, and STAT3 signals, but interestingly enhanced phospho-p53 signal. Overall, our results indicate, for the first time, that Rhy could exert anticancer and anti-metastatic effects through regulation of multiple signaling cascades in hepatocellular carcinoma cells., Competing Interests: The authors declare no conflict of interest.

The pleiotropic transcription factor, signal transducer and activator of transcription 3 (STAT3) is often aberrantly activated in a wide variety of cancers and plays a pivotal role in tumor initiation, promotion and progression. Targeting deregulated STAT3 activation by small molecule inhibitors is generally considered as an important therapeutic strategy. Hence, in the present study, we evaluated the potential of cardamonin (CD), a 2',4'-dihydroxy-6'-methoxychalcone, to modulate STAT3 activation in prostate cancer (PC) cells and found that this chalcone can indeed exhibit significant anticancer effects through negatively regulating STAT3 activation by diverse molecular mechanism(s). CD suppressed STAT3 phosphorylation, nuclear translocation and DNA binding ability in PC cells. Computational modeling revealed that CD can bind directly to the Src Homology 2 domain of STAT3 and also effectively inhibit its dimerization. CD was also found to significantly reduce the migratory/invasive potential of PC cells through the downregulation of various oncogenic proteins. Overall, the data indicates that the potential application of CD as a STAT3 blocker can mitigate both the growth and survival of PC cells.

Condensed-bicyclic 4,6-substituted1,2,4-triazolo-1,3,4-thiadiazole derivatives (CBTT) have been shown to possess a wide spectrum of pharmacological activities. In this study, several novel CBTT derivatives were synthesized and investigated for their possible role as anti-neoplastic agents. The anti-proliferative effect of various CBTT derivatives was analyzed against tumor cell lines by (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) MTT assay. One of the potential CBTT derivative, 5-(3-(2,3-dichlorophenyl)-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazol-6-yl)flurobenzonitrile (DTTF) was found to be the most potent against cervical cancer SiHa cells and exhibited minimal effect against normal cells. Molecular docking analysis indicated that transcription factor NF-κB was one of the potential molecular targets modulated by DTTF. Specifically, the drug blocked the TNFα-induced phosphorylation of upstream IκBα kinase in a time-dependent manner leading to the suppression of NF-κB activation and nuclear translocation. DTTF also potentiated the apoptotic effect of TNFα, as well as significantly inhibited migration and invasion of tumor cells. Overall, these findings indicate a potential novel role and mechanism(s) of action of DTTF as an anticancer agent against diverse malignancies.

Background: Cancer has been one of the leading causes of mortality in this era. Ruta angustifolia L. Pers has been traditionally used as an abortifacient, antihelmintic, emmenagogue and ophthalmic. In Malaysia and Singapore, the local Chinese community used it for the treatment of cancer., Methods: In this study, the methanol and fractionated extracts (hexane, chloroform, ethyl acetate and water) of R. angustifolia were tested for its cytotoxicity using the sulforhodamide (SRB) cytotoxicity assay against HCT-116, A549, Ca Ski and MRC5 cell lines. Chemical isolation was carried out by using the high performance liquid chromatography (HPLC) and the isolated compounds were tested for its cytotoxicity against A549 cell line. Cellular and nuclear morphological changes were observed in the cells using phase contrast microscopy and Hoechst/PI fluorescent staining. The externalisation of phosphatidylserine was observed through FITC-labelling Annexin V/PI assay whilst DNA fragmentation was observed through the TUNEL assay. Other indication of apoptosis occuring through the mitochondrial pathway were the attenuation of mitochondrial membrane potential and increase in ROS production. Activation of caspase 9 and 3 were monitored. Western blot analysis was done to show the expression levels of apoptotic proteins., Results: The chloroform extract (without chlorophyll) exhibited the highest cytotoxic activity with IC 50 of 10.1 ± 0.15 μg/ml against A549 cell line. Further chemical investigation was thus directed to this fraction which led to the isolation of 12 compounds identified as graveoline, psoralen, kokusaginine, methoxysalen, bergapten, arborinine, moskachan B, chalepin, moskachan D, chalepensin, rutamarin and neophytadiene. Among these compounds, chalepin exhibited excellent cytotoxicity against A549 cell line with an IC 50 value of 8.69 ± 2.43 μg/ml (27.64 μM). In western blot analysis, expression of p53, truncated Bid, Bax and Bak while the anti-apoptotic proteins Bcl-2, survivin, XIAP, Bcl-X L ,cFLIP decreased in a time-dependent manner when A549 cells were treated with 36 μg/ml of chalepin. In addition, the level of PARP was found to decrease., Conclusion: Hence these findings indicated that chalepin-induced cell death might involve the intrinsic mitochodrial pathway resulting in the upregulation of pro-apoptotic proteins and downregulation of anti-apoptotic proteins. Thus, chalepin could be an excellent candidate for the development of an anticancer agent.

With conventional anticancer agents for non-small cell lung cancer (NSCLC) reaching therapeutic ceiling, the novel combination using histone deacetylase inhibitor, PXD101 (Belinostat(®)), and CDK inhibitor, CYC202 (Seliciclib(®)), was investigated as an alternative anticancer strategy. At clinically achievable concentration of CYC202 (15 µM), combination therapy resulted in significant reduction in cell proliferation (IC50 = 3.67 ± 0.80 µM, p < 0.05) compared with PXD101 alone (IC50 = 6.56 ± 0.42 µM) in p53 wild-type A549 cells. Significant increase in apoptosis that occurred independently of cell cycle arrest was observed after concurrent treatment. This result was corroborated by greater formation of cleaved caspase-8, caspase-3 and PARP. Up-regulation of p53 and truncated BID protein levels was seen while Mcl-1 and XIAP protein levels were down-regulated upon combined treatment. Further analysis of apoptotic pathways revealed that caspase inhibitors, but not p53 silencing, significantly abrogated the cytotoxic enhancement. Moreover, the enhanced efficacy of this combination was additionally confirmed in p53 null H2444 cells, suggesting the potential of this combination for treatment of NSCLC that are not amenable to effects of conventional p53-inducing agents., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Nimbolide is one of the main components in the leaf extract of Azadirachta indica (A. indica). Accumulating evidence from various in vitro and in vivo studies indicates that nimbolide possesses potent anticancer activity against several types of cancer and also shows potential chemopreventive activity in animal models. The main mechanisms of action of nimbolide include anti-proliferation, induction of apoptosis, inhibition of metastasis and angiogenesis, and modulation of carcinogen-metabolizing enzymes. Although multiple pharmacodynamic (PD) studies have been carried out, nimbolide is still at the infant stage in the drug development pipeline due to the lack of systematic pharmacokinetic (PK) studies and long-term toxicological studies. Preclinical PK and toxicological studies are vital in determining the dosage range to support the safety of nimbolide for first-in-human clinical trials. In this review, we will provide a comprehensive summary for the current status of nimbolide as an anticancer and chemopreventive lead compound, and highlight the importance of systematic preclinical PK and toxicological studies in accelerating the process of application of nimbolide as a therapeutic agent against various malignancies., Competing Interests: Conflicts of Interest: None declared.

Overactivation of PI3K/Akt/mTOR is linked with carcinogenesis and serves a potential molecular therapeutic target in treatment of various cancers. Herein, we report the synthesis of trisubstituted-imidazoles and identified 2-chloro-3-(4, 5-diphenyl-1H-imidazol-2-yl) pyridine (CIP) as lead cytotoxic agent. Naïve Base classifier model of in silico target prediction revealed that CIP targets RAC-beta serine/threonine-protein kinase which comprises the Akt. Furthermore, CIP downregulated the phosphorylation of Akt, PDK and mTOR proteins and decreased expression of cyclin D1, Bcl-2, survivin, VEGF, procaspase-3 and increased cleavage of PARP. In addition, CIP significantly downregulated the CXCL12 induced motility of breast cancer cells and molecular docking calculations revealed that all compounds bind to Akt2 kinase with high docking scores compared to the library of previously reported Akt2 inhibitors. In summary, we report the synthesis and biological evaluation of imidazoles that induce apoptosis in breast cancer cells by negatively regulating PI3K/Akt/mTOR signaling pathway.

Flavokawain C (FKC) is a naturally occurring chalcone which can be found in Kava (Piper methysticum Forst) root. The present study evaluated the effect of FKC on the growth of various human cancer cell lines and the underlying associated mechanisms. FKC showed higher cytotoxic activity against HCT 116 cells in a time- and dose-dependent manner in comparison to other cell lines (MCF-7, HT-29, A549 and CaSki), with minimal toxicity on normal human colon cells. The apoptosis-inducing capability of FKC on HCT 116 cells was evidenced by cell shrinkage, chromatin condensation, DNA fragmentation and increased phosphatidylserine externalization. FKC was found to disrupt mitochondrial membrane potential, resulting in the release of Smac/DIABLO, AIF and cytochrome c into the cytoplasm. Our results also revealed that FKC induced intrinsic and extrinsic apoptosis via upregulation of the levels of pro-apoptotic proteins (Bak) and death receptors (DR5), while downregulation of the levels of anti-apoptotic proteins (XIAP, cIAP-1, c-FlipL, Bcl-xL and survivin), resulting in the activation of caspase-3, -8 and -9 and cleavage of poly(ADP-ribose) polymerase (PARP). FKC was also found to cause endoplasmic reticulum (ER) stress, as suggested by the elevation of GADD153 protein after FKC treatment. After the cells were exposed to FKC (60μM) over 18hrs, there was a substantial increase in the phosphorylation of ERK 1/2. The expression of phosphorylated Akt was also reduced. FKC also caused cell cycle arrest in the S phase in HCT 116 cells in a time- and dose-dependent manner and with accumulation of cells in the sub-G1 phase. This was accompanied by the downregulation of cyclin-dependent kinases (CDK2 and CDK4), consistent with the upregulation of CDK inhibitors (p21Cip1 and p27Kip1), and hypophosphorylation of Rb.

Persistent activation of signal transducer and activator of transcription 3 (STAT3) is one of the characteristic features of renal cell carcinoma (RCC) and often linked to its deregulated proliferation, survival, and angiogenesis. In the present report, we investigated whether zerumbone, a sesquiterpene, exerts its anticancer effect through modulation of STAT3 activation pathway. The pharmacological effect of zerumbone on STAT3 activation, associated protein kinases and phosphatase, and apoptosis was investigated using both RCC cell lines and xenograft mouse model. We observed that zerumbone suppressed STAT3 activation in a dose- and time-dependent manner in RCC cells. The suppression was mediated through the inhibition of activation of upstream kinases c-Src, Janus-activated kinase 1, and Janus-activated kinase 2. Pervanadate treatment reversed zerumbone-induced downregulation of STAT3, suggesting the involvement of a tyrosine phosphatase. Indeed, we found that zerumbone induced the expression of tyrosine phosphatase SHP-1 that correlated with its ability to inhibit STAT3 activation. Interestingly, deletion of SHP-1 gene by siRNA abolished the ability of zerumbone to inhibit STAT3 activation. The inhibition of STAT3 activation by zerumbone also caused the suppression of the gene products involved in proliferation, survival, and angiogenesis. Finally, when administered i.p., zerumbone inhibited STAT3 activation in tumor tissues and the growth of human RCC xenograft tumors in athymic nu/nu mice without any side effects. Overall, our results suggest for the first time that zerumbone is a novel blocker of STAT3 signaling cascade and thus has an enormous potential for the treatment of RCC and other solid tumors., (© 2014 Wiley Periodicals, Inc.)

6-shogaol (6SG), one of active ingredients in ginger (Zingiber officinale), is known to exhibit anti-proliferative, anti-metastatic, and pro-apoptotic activities through a mechanism that is not fully elucidated. Because the aberrant activation of STAT3 and MAPKs have been associated with regulation of proliferation, invasion, and metastasis of tumors, we hypothesized that 6SG modulates the activation of STAT3 and MAPKs activation in tumor cells. We found that 6SG strongly inhibited constitutive phosphorylation of STAT3 through inhibition of the activation of upstream JAK2 and c-Src kinases and nuclear translocation of STAT3 on both MDA-MB231 and DU145 cells. Also, 6SG caused the activation of JNK, p38 MAPK, and ERK. Inhibition of ROS generation by N-acetylcysteine (NAC) significantly prevented 6SG-induced apoptosis. 6SG induced apoptosis as characterized by cleavage of PARP, accumulation of cells in subG1 phase, positive Annexin V binding, down-regulation of STAT3-regulated proteins, and activation of caspase-8, -9, -3 in both MDA-MB231 cells. Compared with other analogues of 6SG, such as 6-gingerol (6G), 8-gingerol (8G), and 10-gingerol (10G), 6SG was found to be the most potent blocker of STAT3 activation. We observed that the administration of 6SG alone significantly suppressed the growth of the tumor. As compared to the vehicle control, 6SG also suppressed the expression of STAT3-regulated gene products such as Bcl-2, Bcl-xL, and Survivin in tumor tissues. Overall, these findings suggest that 6SG can interfere with multiple signaling cascades involved in tumorigenesis and can be used as a potential therapeutic candidate for both the prevention and treatment of cancer., (© 2014 Wiley Periodicals, Inc.)

Persistent activation of signal transducers and activator of transcription 3 (STAT3) has been closely related to growth, survival, proliferation, metastasis, and angiogenesis of various cancer cells, and thus its inhibition can be considered a potential therapeutic strategy. In this study, we investigated the role of bergamottin (BGM) obtained from grapefruit juice in abrogating the constitutive STAT3 activation in multiple myeloma (MM) cells. This suppression was mediated through the inhibition of phosphorylation of Janus-activated kinase (JAK) 1/2 and c-Src. Pervanadate reversed the BGM induced down-regulation of STAT3 activation, suggesting the involvement of a protein tyrosine phosphatase (PTP). Furthermore, BGM induced the expression of the tyrosine phosphatase SHP-1, and gene silencing of the SHP-1 by small interfering RNA abolished the ability of BGM to inhibit STAT3 activation, suggesting a critical role for SHP-1 in the action of BGM. BGM also downregulated the expression of STAT3-regulated gene products such as COX-2, VEGF, cyclin D1, survivin, IAP-1, Bcl-2, and Bcl-xl in MM cells. This correlated with induction of substantial apoptosis as indicated by an increase in the sub-G1 cell population and caspase-3 induced PARP cleavage. Also, this agent significantly potentiated the apoptotic effects of bortezomib and thalidomide in MM cells. Overall, these results suggest that BGM is a novel blocker of STAT3 activation pathway thus may have a potential in therapy of MM and other cancers., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

We have recently reported that β-caryophyllene oxide (CPO) can induce apoptosis, suppress tumor growth, and inhibit metastasis through the suppression of signal transducer and activator of transcription 3, PI3K/AKT/mTOR/S6K1 signaling cascades and ROS-mediated MAPKs activation. In the present study, we found that CPO potentiated the apoptosis induced by tumor necrosis factor α (TNFα) and chemotherapeutic agents, suppressed TNFα-induced tumor cell invasion, all of which are known to require NF-κB activation. We found that TNFα stimulated the expression of gene products involved in anti-apoptosis (IAP1, IAP2, Bcl-2, Bcl-xL, and survivin), proliferation (COX-2, cyclin D1, and c-Myc), invasion (MMP 9 and ICAM-1), and angiogenesis (VEGF) and that CPO treatment suppressed their expression. Because these gene products are also regulated by proinflammatory transcription factor NF-κB, we postulated that CPO may mediate its effects by modulating the NF-κB pathway. We found that CPO blocked both inducible and constitutive NF-κB activation in a wide variety of tumor cells. CPO was also found to inhibit the TNFα-induced degradation of IκBα through the inhibition of activation of IκBα kinase and p65 nuclear translocation and phosphorylation. Interestingly, CPO failed to potentiate the apoptotic effect induced by TNFα in p65 (-/-) cells as compared to the wild-type. Thus, overall, our results indicate that the inhibition of NF-κB is one of major mechanisms by which CPO enhances TNFα-induced apoptosis and suppresses invasion.

Due to narrow therapeutic window of cancer therapeutic agents and the development of resistance against these agents, there is a need to discover novel agents to treat breast cancer. The antitumor activities of thymoquinone (TQ), a compound isolated from Nigella sativa oil, were investigated in breast carcinoma in vitro and in vivo. Cell responses after TQ treatment were assessed by using different assays including MTT assay, annexin V-propidium iodide staining, Mitosox staining and Western blot. The antitumor effect was studied by breast tumor xenograft mouse model, and the tumor tissues were examined by histology and immunohistochemistry. The level of anti-oxidant enzymes/molecules in mouse liver tissues was measured by commercial kits. Here, we show that TQ induced p38 phosphorylation and ROS production in breast cancer cells. These inductions were found to be responsible for TQ's anti-proliferative and pro-apoptotic effects. Moreover, TQ-induced ROS production regulated p38 phosphorylation but not vice versa. TQ treatment was found to suppress the tumor growth and this effect was further enhanced by combination with doxorubicin. TQ also inhibited the protein expression of anti-apoptotic genes, such as XIAP, survivin, Bcl-xL and Bcl-2, in breast cancer cells and breast tumor xenograft. Reduced Ki67 and increased TUNEL staining were observed in TQ-treated tumors. TQ was also found to increase the level of catalase, superoxide dismutase and glutathione in mouse liver tissues. Overall, our results demonstrated that the anti-proliferative and pro-apoptotic effects of TQ in breast cancer are mediated through p38 phosphorylation via ROS generation.

Recombinant tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is currently under clinical trials for cancer, however many tumor cells, including hepatocellular carcinoma (HCC) develop resistance to TRAIL-induced apoptosis. Hence, novel agents that can alleviate TRAIL-induced resistance are urgently needed. In the present report, we investigated the potential of emodin to enhance apoptosis induced by TRAIL in HCC cells. As observed by MTT cytotoxicity assay and the externalization of the membrane phospholipid phosphatidylserine, we found that emodin can significantly potentiate TRAIL-induced apoptosis in HCC cells. When investigated for the mechanism(s), we observed that emodin can downregulate the expression of various cell survival proteins, and induce the cell surface expression of both TRAIL receptors, death receptors (DR) 4 as well as 5. In addition, emodin increased the expression of C/EBP homologous protein (CHOP) in a time-dependent manner. Knockdown of CHOP by siRNA decreased the induction of emodin-induced DR5 expression and apoptosis. Emodin-induced induction of DR5 was mediated through the generation of reactive oxygen species (ROS), as N-acetylcysteine blocked the induction of DR5 and the induction of apoptosis. Also, the knockdown of X-linked inhibitor of apoptosis protein by siRNA significantly reduced the sensitization effect of emodin on TRAIL-induced apoptosis. Overall, our experimental results clearly indicate that emodin can indeed potentiate TRAIL-induced apoptosis through the downregulation of antiapoptotic proteins, increased expression of apoptotic proteins, and ROS mediated upregulation of DR in HCC cells.

Background and Purpose: Aberrant activation of STAT3 is frequently encountered and promotes proliferation, survival, metastasis and angiogenesis in hepatocellular carcinoma (HCC). Here, we have investigated whether emodin mediates its effect through interference with the STAT3 activation pathway in HCC., Experimental Approach: The effect of emodin on STAT3 activation, associated protein kinases and apoptosis was investigated using various HCC cell lines. Additionally, we also used a predictive tumour technology to analyse the effects of emodin . The in vivo effects of emodin were assessed in an orthotopic mouse model of HCC., Key Results: Emodin suppressed STAT3 activation in a dose- and time-dependent manner in HCC cells, mediated by the modulation of activation of upstream kinases c-Src, JAK1 and JAK2. Vanadate treatment reversed emodin-induced down-regulation of STAT3, suggesting the involvement of a tyrosine phosphatase and emodin induced the expression of the tyrosine phosphatase SHP-1 that correlated with the down-regulation of constitutive STAT3 activation. Interestingly, silencing of the SHP-1 gene by siRNA abolished the ability of emodin to inhibit STAT3 activation. Finally, when administered i.p., emodin inhibited the growth of human HCC orthotopic tumours in male athymic nu/nu mice and STAT3 activation in tumour tissues., Conclusions and Implications: Emodin mediated its effects predominantly through inhibition of the STAT3 signalling cascade and thus has a particular potential for the treatment of cancers expressing constitutively activated STAT3., (© 2013 The British Pharmacological Society.)

Constitutive activation of proinflammatory transcription factors such as STAT3 and NF-κB plays a pivotal role in the proliferation and survival of squamous cell carcinoma of the head and neck (HNSCC). Thus, the agents that can modulate deregulated STAT3 and NF-κB activation have a great potential both for the prevention and treatment of HNSCC. In the present report, we investigated the potential effects of garcinol, an active component of Garcinia indica on various inflammatory mediators involved in HNSCC progression using cell lines and xenograft mouse model. We found that garcinol inhibited constitutively activated STAT3 in HNSCC cells in a time- and dose-dependent manner, which correlated with the suppression of the upstream kinases (c-Src, JAK1, and JAK2) in HNSCC cells. Also, we noticed that the generation of reactive oxygen species is involved in STAT3 inhibitory effect of garcinol. Furthermore, garcinol exhibited an inhibitory effect on the constitutive NF-κB activation, mediated through the suppression of TGF-β-activated kinase 1 (TAK1) and inhibitor of IκB kinase (IKK) activation in HNSCC cells. Garcinol also downregulated the expression of various gene products involved in proliferation, survival, and angiogenesis that led to the reduction of cell viability and induction of apoptosis in HNSCC cells. When administered intraperitoneally, garcinol inhibited the growth of human HNSCC xenograft tumors in male athymic nu/nu mice. Overall, our results suggest for the first time that garcinol mediates its antitumor effects in HNSCC cells and mouse model through the suppression of multiple proinflammatory cascades.

Background: Akt/mTOR/S6K1 signaling cascades play an important role both in the survival and proliferation of tumor cells., Methods: In the present study, we investigated the effects of embelin (EB), identified primarily from the Embelia ribes plant, on the Akt/mTOR/S6K1 activation, associated gene products, cellular proliferation, and apoptosis in human prostate cancer cells., Results: EB exerted significant cytotoxic and suppressive effects on Akt and mTOR activation against androgen-independent PC-3 cells as compared to androgen-dependent LNCaP cells. Moreover, EB suppressed the constitutive activation of Akt/mTOR/S6K1 signaling cascade, which correlated with the induction of apoptosis as characterized by accumulation of cells in subG1 phase, positive Annexin V binding, down-regulation of anti-apoptotic (Bcl-2, Bcl-xL, survivin, IAP-1, and IAP-2) and proliferative (cyclin D1) proteins, activation of caspase-3, and cleavage of PARP. We also observed that EB can significantly enhance the apoptotic effects of a specific pharmacological Akt inhibitor when used in combination and also caused broad inhibition of all the three kinases in Akt/mTOR/S6K1 signaling axis in PC-3 cells., Conclusions: EB inhibits multiple signaling cascades involved in tumorigenesis and can be used as a potential therapeutic candidate for both the prevention and treatment of prostate cancer., (Copyright © 2012 Wiley Periodicals, Inc.)

Gastric cancer (GC) is a lethal malignancy and the second most common cause of cancer-related deaths. Although treatment options such as chemotherapy, radiotherapy, and surgery have led to a decline in the mortality rate due to GC, chemoresistance remains as one of the major causes for poor prognosis and high recurrence rate. In this study, we investigated the potential effects of isorhamnetin (IH), a 3'-O-methylated metabolite of quercetin on the peroxisome proliferator-activated receptor γ (PPAR-γ) signaling cascade using proteomics technology platform, GC cell lines, and xenograft mice model. We observed that IH exerted a strong antiproliferative effect and increased cytotoxicity in combination with chemotherapeutic drugs. IH also inhibited the migratory/invasive properties of GC cells, which could be reversed in the presence of PPAR-γ inhibitor. We found that IH increased PPAR-γ activity and modulated the expression of PPAR-γ regulated genes in GC cells. Also, the increase in PPAR-γ activity was reversed in the presence of PPAR-γ-specific inhibitor and a mutated PPAR-γ dominant negative plasmid, supporting our hypothesis that IH can act as a ligand of PPAR-γ. Using molecular docking analysis, we demonstrate that IH formed interactions with seven polar residues and six nonpolar residues within the ligand-binding pocket of PPAR-γ that are reported to be critical for its activity and could competitively bind to PPAR-γ. IH significantly increased the expression of PPAR-γ in tumor tissues obtained from xenograft model of GC. Overall, our findings clearly indicate that antitumor effects of IH may be mediated through modulation of the PPAR-γ activation pathway in GC.

Cumulative evidences(s) have established that the constitutive activation of STAT3 plays a pivotal role in the proliferation, survival, metastasis, and angiogenesis and thus can contribute directly to the pathogenesis of hepatocellular carcinoma (HCC). Thus, novel agents that can inhibit STAT3 activation have potential for both prevention and treatment of HCCs. The effect of celastrol on STAT3 activation, associated protein kinases, STAT3-regulated gene products, cellular proliferation, and apoptosis was investigated. The in vivo effect of celastrol on the growth of human HCC xenograft tumors in athymic nu/nu mice was also examined. We observed that celastrol inhibited both constitutive and inducible STAT3 activation, and the suppression was mediated through the inhibition of activation of upstream kinases c-Src, as well as Janus-activated kinase-1 and -2. Vanadate treatment reversed the celastrol-induced modulation of STAT3, suggesting the involvement of a tyrosine phosphatase. The inhibition of STAT3 activation by celastrol led to the suppression of various gene products involved in proliferation, survival, and angiogenesis. Celastrol also inhibited the proliferation and induced apoptosis in HCC cells. Finally, when administered intraperitoneally, celastrol inhibited STAT3 activation in tumor tissues and the growth of human HCC xenograft tumors in athymic nu/nu mice without any side effects. Overall, our results suggest for the first time that celastrol exerts its antiproliferative and proapoptotic effects through suppression of STAT3 signaling in HCC both in vitro and in vivo., (2012 AACR)

Both PI3K/AKT/mTOR/S6K1 and mitogen activated protein kinase (MAPK) signaling cascades play an important role in cell proliferation, survival, angiogenesis, and metastasis of tumor cells. In the present report, we investigated the effects of β-caryophyllene oxide (CPO), a sesquiterpene isolated from essential oils of medicinal plants such as guava (Psidium guajava), oregano (Origanum vulgare L.), cinnamon (Cinnamomum spp.) clove (Eugenia caryophyllata), and black pepper (Piper nigrum L.) on the PI3K/AKT/mTOR/S6K1 and MAPK activation pathways in human prostate and breast cancer cells. We found that CPO not only inhibited the constitutive activation of PI3K/AKT/mTOR/S6K1 signaling cascade; but also caused the activation of ERK, JNK, and p38 MAPK in tumor cells. CPO induced increased reactive oxygen species (ROS) generation from mitochondria, which is associated with the induction of apoptosis as characterized by positive Annexin V binding and TUNEL staining, loss of mitochondrial membrane potential, release of cytochrome c, activation of caspase-3, and cleavage of PARP. Inhibition of ROS generation by N-acetylcysteine (NAC) significantly prevented CPO-induced apoptosis. Subsequently, CPO also down-regulated the expression of various downstream gene products that mediate cell proliferation (cyclin D1), survival (bcl-2, bcl-xL, survivin, IAP-1, and IAP-2), metastasis (COX-2), angiogenesis (VEGF), and increased the expression of p53 and p21. Interestingly, we also observed that CPO can significantly potentiate the apoptotic effects of various pharmacological PI3K/AKT inhibitors when employed in combination in tumor cells. Overall, these findings suggest that CPO can interfere with multiple signaling cascades involved in tumorigenesis and used as a potential therapeutic candidate for both the prevention and treatment of cancer., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Celastrol, a plant triterpene has attracted great interest recently, especially for its potential anti-inflammatory and anti-cancer activities. In the present report, we investigated the effect of celastrol on proliferation of various cancer cell lines. The mechanism, by which this triterpene exerts its apoptotic effects, was also examined in detail. We found that celastrol inhibited the proliferation of wide variety of human tumor cell types including multiple myeloma, hepatocellular carcinoma, gastric cancer, prostate cancer, renal cell carcinoma, head and neck carcinoma, non-small cell lung carcinoma, melanoma, glioma, and breast cancer with concentrations as low as 1 μM. Growth inhibitory effects of celastrol correlated with a decrease in the levels of cyclin D1 and cyclin E, but concomitant increase in the levels of p21 and p27. The apoptosis induced by celastrol was indicated by the activation of caspase-8, bid cleavage, caspase-9 activation, caspase-3 activation, PARP cleavage and through the down regulation of anti-apoptototic proteins. The apoptotic effects of celastrol were preceded by activation of JNK and down-regulation of Akt activation. JNK was needed for celastrol-induced apoptosis, and inhibition of JNK by pharmacological inhibitor abolished the apoptotic effects. Overall, our results indicate that celastrol can inhibit cell proliferation and induce apoptosis through the activation of JNK, suppression of Akt, and down-regulation of anti-apoptotic protein expression.

Background and Purpose: Activation of signal transducer and activator of transcription 3 (STAT3) play a critical role in the survival, proliferation, angiogenesis and chemoresistance of tumour cells. Thus, agents that suppress STAT3 phosphorylation have potential as cancer therapies. In the present study, we investigated whether the apoptotic, antiproliferative and chemosensitizing effects of γ-tocotrienol are associated with its ability to suppress STAT3 activation in hepatocellular carcinoma (HCC)., Experimental Approach: The effect of γ-tocotrienol on STAT3 activation, associated protein kinases and phosphatase, STAT3-regulated gene products, cellular proliferation and apoptosis in HCC cells was investigated., Key Results: γ-Tocotrienol inhibited both the constitutive and inducible activation of STAT3 with minimum effect on STAT5. γ-Tocotrienol also inhibited the activation of Src, JAK1 and JAK2 implicated in STAT3 activation. Pervanadate reversed the γ-tocotrienol-induced down-regulation of STAT3, suggesting the involvement of a protein tyrosine phosphatase. Indeed, we found that γ-tocotrienol induced the expression of the tyrosine phosphatase SHP-1 and deletion of the SHP-1 gene by small interfering RNA abolished the ability of γ-tocotrienol to inhibit STAT3 activation. γ-Tocotrienol also down-regulated the expression of STAT3-regulated gene products, including cyclin D1, Bcl-2, Bcl-xL, survivin, Mcl-1 and vascular endothelial growth factor. Finally, γ-tocotrienol inhibited proliferation, induced apoptosis and significantly potentiated the apoptotic effects of chemotherapeutic drugs (paclitaxel and doxorubicin) used for the treatment of HCC., Conclusions and Implications: Overall, these results suggest that γ-tocotrienol is a novel blocker of the STAT3 activation pathway, with a potential role in future therapies for HCC and other cancers., (© 2011 The Authors. British Journal of Pharmacology © 2011 The British Pharmacological Society.)

Panax ginseng has been reported to have cancer-preventive properties and, through anti-inflammatory, antioxidant, and pro-apoptotic mechanisms, to influence gene expression. However, the comparison of Korean white ginseng (WG) and red ginseng (RG) in their apoptotic effects and the identification of the selective cellular uptake of the ginsenosides in human breast cancer cells have not yet been fully understood. In the present study, the relative nonpolar and protopanaxadiol (PPD) class ginsenosides exhibited more cytotoxic and efficient cellular uptake on MCF-7 cells compared with the relative polar and protopanaxatriol (PPT) class compounds. PPD class ginsenosides were present in RG in a 2.5 times higher concentration as compared to WG, while PPT class ginsenosides were only present in WG. Thus, RG exerted more potent cytotoxicity than WG against MCF-7 and MDA-MB231 cells. RG also increased the sub-G1 DNA contents of the cell cycle and Annexin V-positive apoptotic bodies undergoing apoptosis through the caspase-3 activation in MCF-7 cells. In addition, RG downregulated the proliferative and anti-apoptotic gene products and potentiated paclitaxel-induced apoptosis in MCF-7 cells. Overall, RG contained a higher concentration of PPD class ginsenosides as compared to WG; the greater cellular uptake of PPD resulted in more substantial antiproliferative activity in human breast cancer cells., (© Georg Thieme Verlag KG Stuttgart · New York.)

Cancer is one of the leading causes of death in the United States and around the world. Most modern drug-targeted therapies, besides being enormously expensive, are associated with serious side effects and morbidity. Still, the search continues for an ideal treatment that has minimal side effects and is cost-effective. Indeed, the design and development of chemopreventive agents that act on specific and/or multiple molecular and cellular targets is gaining support as a rational approach to prevent and treat cancer. We present evidence on numerous dietary agents identified from fruits and vegetables that act on multiple signal transduction and apoptotic cascades in various tumor cells and animal models. Some of the most interesting and well documented are turmeric (curcumin), resveratrol, silymarin, EGCG, and genistein. This review will provide an insight on the cellular and molecular mechanism(s) by which dietary agents modulate multiple signaling and apoptotic pathways in tumor cells and elucidate the role of these agents in both prevention and treatment of cancer., (Copyright © 2011, Taylor & Francis Group, LLC)

Background and Purpose: Constitutive activation of the signal transducer and activator of transcription 3 (STAT3) pathway is frequently encountered in several human cancers including multiple myeloma (MM). Thus, agents that suppress STAT3 phosphorylation have a potential for treatment of MM. In the present report, we investigated whether thymoquinone (TQ), the main component isolated from the medicinal plant Nigella sativa, modulated the STAT3 signalling pathway in MM cells., Experimental Approach: The effect of TQ on both constitutive and IL-6-induced STAT3 activation, associated protein kinases, STAT3-regulated gene products involved in proliferation, survival and angiogenesis, cellular proliferation and apoptosis in MM cells, was investigated., Key Results: We found that TQ inhibited both constitutive and IL-6-inducible STAT3 phosphorylation which correlated with the inhibition of c-Src and JAK2 activation. Vanadate reversed the TQ-induced down-regulation of STAT3 activation, suggesting the involvement of a protein tyrosine phosphatase. Indeed, we found that TQ can induce the expression of Src homology-2 phosphatase 2 that correlated with suppression of STAT3 activation. TQ also down-regulated the expression of STAT3-regulated gene products, such as cyclin D1, Bcl-2, Bcl-xL, survivin, Mcl-1 and vascular endothelial growth factor. Finally, TQ induced the accumulation of cells in sub-G1 phase, inhibited proliferation and induced apoptosis, as indicated by poly ADP ribose polymerase cleavage. TQ also significantly potentiated the apoptotic effects of thalidomide and bortezomib in MM cells., Conclusions and Implications: Our study has identified STAT3 signalling as a target of TQ and has thus raised its potential application in the prevention and treatment of MM and other cancers.

The activation of signal transducer and activator of transcription 3 (STAT3) has been linked with the proliferation, survival, invasion, and angiogenesis of a variety of human cancer cells, including hepatocellular carcinoma (HCC). Agents that can suppress STAT3 activation have potential for the prevention and treatment of HCC. In this study, we tested an agent, beta-escin, for its ability to suppress STAT3 activation. We found that beta-escin, a pentacyclic triterpenoid, inhibited both constitutive and interleukin-6-inducible STAT3 activation in a dose- and time-dependent manner in HCC cells. The suppression was mediated through the inhibition of activation of upstream kinases c-Src, Janus-activated kinase 1, and Janus-activated kinase 2. Vanadate treatment reversed the beta-escin-induced down-regulation of STAT3, suggesting the involvement of a tyrosine phosphatase. Indeed, we found that beta-escin induced the expression of tyrosine phosphatase Src homology phosphatase 1 that correlated with the down-regulation of constitutive STAT3 activation. beta-Escin also down-regulated the expression of STAT3-regulated gene products, such as cyclin D1, Bcl-2, Bcl-xL, survivin, Mcl-1, and vascular endothelial growth factor. Finally, beta-escin inhibited proliferation and also substantially potentiated the apoptotic effects of paclitaxel and doxorubicin in HCC cells. Overall, these results suggest that beta-escin is a novel blocker of STAT3 activation that may have potential in the suppression of proliferation and chemosensitization in HCC.

Curcumin (diferuloylmethane), a yellow pigment in turmeric, has been shown to inhibit the activation of nuclear factor-kappaB (NF-kappaB), a transcription factor closely linked to chemoresistance in multiple myeloma cells. Whether curcumin can overcome chemoresistance and enhance the activity of thalidomide and bortezomib, used to treat patients with multiple myeloma, was investigated in vitro and in xenograft model in nude mice. Our results show that curcumin inhibited the proliferation of human multiple myeloma cells regardless of their sensitivity to dexamethasone, doxorubicin, or melphalan. Curcumin also potentiated the apoptotic effects of thalidomide and bortezomib by down-regulating the constitutive activation of NF-kappaB and Akt, and this correlated with the suppression of NF-kappaB-regulated gene products, including cyclin D1, Bcl-xL, Bcl-2, TRAF1, cIAP-1, XIAP, survivin, and vascular endothelial growth factor. Furthermore, in a nude mice model, we found that curcumin potentiated the antitumor effects of bortezomib (P<0.001, vehicle versus bortezomib+curcumin; P<0.001, bortezomib versus bortezomib+curcumin), and this correlated with suppression of Ki-67 (P<0.001 versus control), CD31 (P<0.001 versus vehicle), and vascular endothelial growth factor (P<0.001 versus vehicle) expression. Collectively, our results suggest that curcumin overcomes chemoresistance and sensitizes multiple myeloma cells to thalidomide and bortezomib by down-regulating NF-kappaB and NF-kappaB-regulated gene products.

Because the phosphatidylinositol-3-kinase-AKT pathway is emerging as an important regulator of tumor cell survival, inhibitors of this pathway have enormous potential in cancer treatment. A specific inhibitor of AKT, [d-3-deoxy-2-O-methyl-myo-inositol-1-[(R)-2-methoxy-3-(octadecyloxy)propyl hydrogen phosphate]] (SH-5) has been recently synthesized, but little is known about its effects on cytokine signaling. We found that SH-5 potentiated the apoptosis induced by tumor necrosis factor (TNF), as indicated by intracellular esterase staining, annexin V staining, and caspase-3 activation. This effect of SH-5 correlated with downregulation of various gene products that mediate cell survival, proliferation, metastasis, and invasion, all known to be regulated by NF-kappaB. SH-5 also blocked NF-kappaB activation induced by TNF-alpha, lipopolysaccharide, phorbol ester, and cigarette smoke but not that activated by hydrogen peroxide and RANK ligand, indicating differential requirement of AKT. Inhibition of NF-kappaB correlated with abrogation of phosphorylation and degradation of IkappaBalpha through the inhibition of activation of IkappaBalpha kinase (IKK). This led to suppression of the phosphorylation and translocation of p65 and also of NF-kappaB reporter activity induced by TNFR1, TRADD, TRAF2, NIK, and IKKbeta but not that induced by p65 transfection. Thus, our results clearly demonstrate that inhibition of AKT leads to potentiation of apoptosis through modulation of NF-kappaB signaling.

Berberine, an isoquinoline alkaloid derived from a plant used traditionally in Chinese and Ayurvedic medicine, has been reported to exhibit chemopreventive and anti-inflammatory activities through unknown mechanism. Because of the critical role of the transcription factor nuclear factor-kappaB (NF-kappaB) in these processes, we investigated the effect of berberine on this pathway. We found that berberine suppressed NF-kappaB activation induced by various inflammatory agents and carcinogens. This alkaloid also suppressed constitutive NF-kappaB activation found in certain tumor cells. Suppression of NF-kappaB activation occurred through the inhibition of phosphorylation and degradation of IkappaBalpha by the inhibition of IkappaB kinase (IKK) activation, leading to suppression of phosphorylation and nuclear translocation of p65, and finally to inhibition of NF-kappaB reporter activity. Inhibition of IKK by berbeine was direct and could be reversed by reducing agents. Site-specific mutagenesis suggested the involvement of cysteine residue 179 in IKK. Berberine also suppressed the expression of NF-kappaB-regulated gene products involved in antiapoptosis (Bcl-xL, Survivin, IAP1, IAP2, and cFLIP), proliferation (cyclin D1), inflammation (cyclooxygenase-2), and invasion (matrix metalloproteinase-9). Suppression of antiapoptotic gene products correlated with enhancement of apoptosis induced by tumor necrosis factor (TNF)-alpha and chemotherapeutic agents and with inhibition of TNF-induced cellular invasion. Overall, our results indicate that chemopreventive, apoptotic, and anti-inflammatory activities displayed by berberine may be mediated in part through the suppression of the NF-kappaB activation pathway. This may provide the molecular basis for the ability of berberine to act as an anticancer and anti-inflammatory agent.

The molecular mechanisms of pro-apoptotic effects of human-derived Lactobacillus reuteri ATCC PTA 6475 were investigated in this study. L. reuteri secretes factors that potentiate apoptosis in myeloid leukemia-derived cells induced by tumour necrosis factor (TNF), as indicated by intracellular esterase activity, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labelling assays and poly (ADP-ribose) polymerase cleavage. L. reuteri downregulated nuclear factor-kappaB (NF-kappaB)-dependent gene products that mediate cell proliferation (Cox-2, cyclin D1) and cell survival (Bcl-2, Bcl-xL). L. reuteri suppressed TNF-induced NF-kappaB activation, including NF-kappaB-dependent reporter gene expression in a dose-and time-dependent manner. L. reuteri stabilized degradation of IkappaBalpha and inhibited nuclear translocation of p65 (RelA). Although phosphorylation of IkappaBalpha was not affected, subsequent polyubiquitination necessary for regulated IkappaBalpha degradation was abrogated by L. reuteri. In addition, L. reuteri promoted apoptosis by enhancing mitogen-activated protein kinase (MAPK) activities including c-Jun N-terminal kinase and p38 MAPK. In contrast, L. reuteri suppressed extracellular signal-regulated kinases 1/2 in TNF-activated myeloid cells. L. reuteri may regulate cell proliferation by promoting apoptosis of activated immune cells via inhibition of IkappaBalpha ubiquitination and enhancing pro-apoptotic MAPK signalling. An improved understanding of L. reuteri-mediated effects on apoptotic signalling pathways may facilitate development of future probiotics-based regimens for prevention of colorectal cancer and inflammatory bowel disease.

Pinitol (3-O-methyl-chiroinositol), a component of traditional Ayurvedic medicine (talisapatra), has been shown to exhibit anti-inflammatory and antidiabetic activities through undefined mechanisms. Because the transcription factor nuclear factor-kappaB (NF-kappaB) has been linked with inflammatory diseases, including insulin resistance, we hypothesized that pinitol must mediate its effects through modulation of NF-kappaB activation pathway. We found that pinitol suppressed NF-kappaB activation induced by inflammatory stimuli and carcinogens. This suppression was not specific to cell type. Besides inducible, pinitol also abrogated constitutive NF-kappaB activation noted in most tumor cells. The suppression of NF-kappaB activation by pinitol occurred through inhibition of the activation of IkappaBalpha kinase, leading to sequential suppression of IkappaBalpha phosphorylation, IkappaBalpha degradation, p65 phosphorylation, p65 nuclear translocation, and NF-kappaB-dependent reporter gene expression. Pinitol also suppressed the NF-kappaB reporter activity induced by tumor necrosis factor receptor (TNFR)-1, TNFR-associated death domain, TNFR-associated factor-2, transforming growth factor-beta-activated kinase-1 (TAK-1)/TAK1-binding protein-1, and IkappaBalpha kinase but not that induced by p65. The inhibition of NF-kappaB activation thereby led to down-regulation of gene products involved in inflammation (cyclooxygenase-2), proliferation (cyclin D1 and c-myc), invasion (matrix metalloproteinase-9), angiogenesis (vascular endothelial growth factor), and cell survival (cIAP1, cIAP2, X-linked inhibitor apoptosis protein, Bcl-2, and Bcl-xL). Suppression of these gene products by pinitol enhanced the apoptosis induced by TNF and chemotherapeutic agents and suppressed TNF-induced cellular invasion. Our results show that pinitol inhibits the NF-kappaB activation pathway, which may explain its ability to suppress inflammatory cellular responses.

Thymoquinone (TQ), derived from the medicinal plant Nigella sativa, exhibits antiinflammatory and anticancer activities through mechanism(s) that is not fully understood. Because numerous effects modulated by TQ can be linked to interference with the nuclear factor-kappaB (NF-kappa B) signaling, we investigated in detail the effect of this quinone on NF-kappa B pathway. As examined by DNA binding, we found that TQ suppressed tumor necrosis factor-induced NF-kappa B activation in a dose- and time-dependent manner and inhibited NF-kappaB activation induced by various carcinogens and inflammatory stimuli. The suppression of NF-kappaB activation correlated with sequential inhibition of the activation of I kappa B alpha kinase, I kappa B alpha phosphorylation, I kappa B alpha degradation, p65 phosphorylation, p65 nuclear translocation, and the NF-kappa B-dependent reporter gene expression. TQ specifically suppressed the direct binding of nuclear p65 and recombinant p65 to the DNA, and this binding was reversed by DTT. However, TQ did not inhibit p65 binding to DNA when cells were transfected with the p65 plasmid containing cysteine residue 38 mutated to serine. TQ also down-regulated the expression of NF-kappa B-regulated antiapoptotic (IAP1, IAP2, XIAP Bcl-2, Bcl-xL, and survivin), proliferative (cyclin D1, cyclooxygenase-2, and c-Myc), and angiogenic (matrix metalloproteinase-9 and vascular endothelial growth factor) gene products. This led to potentiation of apoptosis induced by tumor necrosis factor and chemotherapeutic agents. Overall, our results indicate that the anticancer and antiinflammatory activities previously assigned to TQ may be mediated in part through the suppression of the NF-kappa B activation pathway, as shown here, and thus may have potential in treatment of myeloid leukemia and other cancers.

Although flavopiridol, a semisynthetic flavone, was initially thought to be a specific inhibitor of cyclin-dependent kinases, it has now been shown that flavopiridol mediates antitumor responses through mechanism(s) yet to be defined. We have shown previously that flavopiridol abrogates tumor necrosis factor (TNF)-induced nuclear factor-kappaB (NF-kappaB) activation. In this report, we examined whether this flavone affects other cellular responses activated by TNF. TNF is a potent inducer of activator protein-1 (AP-1), and flavopiridol abrogated this activation in a dose- and time-dependent manner. Flavopiridol also suppressed AP-1 activation induced by various carcinogens and inflammatory stimuli. When examined for its effect on other signaling pathways, flavopiridol inhibited TNF-induced activation of various mitogen-activated protein kinases, including c-Jun NH(2)-terminal kinase (JNK), p38 mitogen-activated protein kinase (MAPK), and p44/p42 MAPK. It is noteworthy that this flavone also suppressed TNF-induced activation of Akt, a cell survival kinase, and expression of various antiapoptotic proteins, such as IAP-1, IAP-2, XIAP, Bcl-2, Bcl-xL, and TRAF-1. Flavopiridol also inhibited the TNF-induced induction of intercellular adhesion molecule-1, c-Myc, and c-Fos, all known to mediate tumorigenesis. Moreover, TNF-induced apoptosis was enhanced by flavopiridol through activation of the bid-cytochrome-caspase-9-caspase-3 pathway. Overall, our results clearly suggest that flavopiridol interferes with the TNF cell-signaling pathway, leading to suppression of antiapoptotic mechanisms and enhancement of apoptosis.

We recently found that simvastatin can modulate the nuclear factor-kappaB (NF-kappaB) activation pathway, but whether other statins have similar effects to those of simvastatin is unknown. Therefore, we evaluated the effect six different statins on TNF-induced NF-kappaB activation in human myeloid leukemia cells. We then determined whether the combination of statins and standard chemotherapeutic agents could overcome chemoresistance and augment apoptosis. Of the six statins evaluated, only the natural statins (simvastatin, mevastatin, lovastatin, and pravastatin), not the synthetic statins (fluvastatin and atorvastatin), inhibited TNF-induced NF-kappaB activation. Simvastatin suppressed the NF-kappaB activation and potentiated the apoptosis induced by doxorubicin, paclitaxel, and 5-fluorouracil. These results suggest that different statins behave differently from one another and that they may be useful in overcoming chemoresistance.

Almost 19 members of the tumor necrosis factor (TNF) superfamily have been identified that interact with 29 different receptors. Whether these receptors communicate with each other is not understood. Recently, we have shown that receptor activator of NF-kappaB ligand signaling is modulated by genetic deletion of the TNF receptor. In the current report, we investigated the possibility of a cross-talk between Fas and TNF-alpha signaling pathway in macrophage cell lines derived from wild-type (WT) mice and from mice with genetic deletion of the type 1 TNF receptor (p60(-/-)), the type 2 TNF receptor (p80(-/-)), or both receptors (p60(-/-)p80(-/-)). We found that the macrophages expressing TNF receptors were highly sensitive to apoptosis induced by anti-Fas. The genetic deletion of TNF receptors, however, made the cells resistance to anti-Fas-induced apoptosis. Anti-Fas induced activation of caspase-3 and PARP cleavage in WT cells but not in TNF receptor-deleted cells. This difference was found to be independent of the expression of Fas, Fas-associated protein with death domain (FADD) or TNF receptor-associated death domain (TRADD). We found that anti-Fas induced recruitment of TNFR1 into Fas-complex. We also found that TRADD, which mediates TNF signaling, was constitutively bound to Fas receptor in TNF receptor-deleted cells but not in wild-type cells. Transient transfection of TNFR1 in TNFR1-deleted cells sensitized them to anti-Fas-induced apoptosis. Overall our results demonstrate that Fas signaling is modulated by the TNF receptors and thus provide the evidence of cross-talk between the receptors of two cytokines.

Salinosporamide A (also called NPI-0052), recently identified from the marine bacterium Salinispora tropica, is a potent inhibitor of 20S proteasome and exhibits therapeutic potential against a wide variety of tumors through a poorly understood mechanism. Here we demonstrate that salinosporamide A potentiated the apoptosis induced by tumor necrosis factor alpha (TNF), bortezomib, and thalidomide, and this correlated with down-regulation of gene products that mediate cell proliferation (cyclin D1, cyclooxygenase-2 [COX-2], and c-Myc), cell survival (Bcl-2, Bcl-xL, cFLIP, TRAF1, IAP1, IAP2, and survivin), invasion (matrix metallopro-teinase-9 [MMP-9] and ICAM-1), and angiogenesis (vascular endothelial growth factor [VEGF]). Salinosporamide A also suppressed TNF-induced tumor cell invasion and receptor activator of nuclear factor kappaB ligand (RANKL)-induced osteoclastogenesis. We also found that it suppressed both constitutive and inducible NF-kappaB activation. Compared with bortezomib, MG-132, N-acetyl-leucyl-leucyl-norleucinal (ALLN), and lactacystin, salinosporamide A was found to be the most potent suppressor of NF-kappaB activation. Further studies showed that salinosporamide A inhibited TNF-induced inhibitory subunit of NF-kappaB alpha (IkappaBalpha) degradation, nuclear translocation of p65, and NF-kappaB-dependent reporter gene expression but had no effect on IkappaBalpha kinase activation, IkappaBalpha phosphorylation, or IkappaBalpha ubiquitination. Thus, overall, our results indicate that salinosporamide A enhances apoptosis, suppresses osteoclastogenesis, and inhibits invasion through suppression of the NF-kappaB pathway.

Extensive research within the past half-century has indicated that curcumin (diferuloylmethane), a yellow pigment in curry powder, exhibits antioxidant, anti-inflammatory, and proapoptotic activities. We investigated whether the anti-inflammatory and proapoptotic activities assigned to curcumin are mediated through its prooxidant/antioxidant mechanism. We found that TNF-mediated NF-kappaB activation was inhibited by curcumin; and glutathione reversed the inhibition. Similarly, suppression of TNF-induced AKT activation by curcumin was also abrogated by glutathione. The reducing agent also counteracted the inhibitory effects of curcumin on TNF-induced NF-kappaB-regulated antiapoptotic (Bcl-2, Bcl-xL, IAP1), proliferative (cyclin D1), and proinflammatory (COX-2, iNOS, and MMP-9) gene products. The suppression of TNF-induced AP-1 activation by curcumin was also reversed by glutathione. Also, the direct proapoptotic effects of curcumin were inhibited by glutathione and potentiated by depletion of intracellular glutathione by buthionine sulfoximine. Moreover, curcumin induced the production of reactive oxygen species and modulated intracellular GSH levels. Quenchers of hydroxyl radicals, however, were ineffective in inhibiting curcumin-mediated NF-kappaB suppression. Further, N-acetylcysteine partially reversed the effect of curcumin. Based on these results we conclude that curcumin mediates its apoptotic and anti-inflammatory activities through modulation of the redox status of the cell.

MAPK kinase 4 (MKK4) is a dual-specificity kinase that activates both JNK and p38 MAPK. However, the mechanism by which MKK4 regulates TNF-induced apoptosis is not fully understood. Therefore, we used fibroblasts derived from MKK4 gene-deleted (MKK4-KO) mice to determine the role of this kinase in TNF signaling. We found that when compared with the wild-type cells, deletion of MKK4 gene enhanced TNF-induced apoptosis, and this correlated with down-regulation of TNF-induced cell-proliferative (COX-2 and cyclin D1) and antiapoptotic (survivin, IAP1, XIAP, Bcl-2, Bcl-x(L), and cFLIP) gene products, all regulated by NF-kappaB. Indeed we found that TNF-induced NF-kappaB activation was abrogated in MKK4 gene-deleted cells, as determined by DNA binding. Further investigation revealed that TNF-induced I kappaB alpha kinase activation, I kappaB alpha phosphorylation, I kappaB alpha degradation, and p65 nuclear translocation were all suppressed in MKK4-KO cells. NF-kappaB reporter assay revealed that NF-kappaB activation induced by TNF, TNFR1, TRADD, TRAF2, NIK, and I kappaB alpha kinase was modulated in gene-deleted cells. Overall, our results indicate that MKK4 plays a central role in TNF-induced apoptosis through the regulation of NF-kappaB-regulated gene products.

Guggulsterone is a plant polyphenol traditionally used to treat obesity, diabetes, hyperlipidemia, atherosclerosis, and osteoarthritis, possibly through an anti-inflammatory mechanism. Whether this steroid has any role in cancer is not known. In this study, we found that guggulsterone inhibits the proliferation of wide variety of human tumor cell types including leukemia, head and neck carcinoma, multiple myeloma, lung carcinoma, melanoma, breast carcinoma, and ovarian carcinoma. Guggulsterone also inhibited the proliferation of drug-resistant cancer cells (e.g., gleevac-resistant leukemia, dexamethasone-resistant multiple myeloma, and doxorubicin-resistant breast cancer cells). Guggulsterone suppressed the proliferation of cells through inhibition of DNA synthesis, producing cell cycle arrest in S-phase, and this arrest correlated with a decrease in the levels of cyclin D1 and cdc2 and a concomitant increase in the levels of cyclin-dependent kinase inhibitor p21 and p27. Guggulsterone-induced apoptosis as indicated by increase in the number of Annexin V- and TUNEL-positive cells, through the downregulation of anti-apoptototic products. The apoptosis induced by guggulsterone was also indicated by the activation of caspase-8, bid cleavage, cytochrome c release, caspase-9 activation, caspase-3 activation, and PARP cleavage. The apoptotic effects of guggulsterone were preceded by activation of JNK and downregulation of Akt activity. JNK was needed for guggulsterone-induced apoptosis, inasmuch as inhibition of JNK by pharmacological inhibitors or by genetic deletion of MKK4 (activator of JNK) abolished the activity. Overall, our results indicate that guggulsterone can inhibit cell proliferation and induce apoptosis through the activation of JNK, suppression of Akt, and downregulation of antiapoptotic protein expression.

Purpose: Morin is a flavone that exhibits antiproliferative, antitumor, and anti-inflammatory effects through a mechanism that is not well understood. Because of the role of transcription factor nuclear factor-kappaB (NF-kappaB) in the control of cell survival, proliferation, tumorigenesis, and inflammation, we postulated that morin mediates its effects by modulating NF-kappaB activation., Experimental Design: We investigated the effect of morin on NF-kappaB pathway activated by inflammatory agents, carcinogens, and tumor promoters. The effect of this flavone on expression of NF-kappaB-regulated gene products involved in cell survival, proliferation, and invasion was also examined., Results: We showed by DNA-binding assay that NF-kappaB activation induced by tumor necrosis factor (TNF), phorbol 12-myristate 13-acetate, lipopolysaccharide, ceramide, interleukin-1, and H(2)O(2) was suppressed by morin; the suppression was not cell type specific. The suppression of NF-kappaB by morin was mediated through inhibition of IkappaBalpha (inhibitory subunit of NF-kappaB) kinase, leading to suppression of phosphorylation and degradation of IkappaBalpha and consequent p65 nuclear translocation. Morin also inhibited the NF-kappaB-dependent reporter gene expression activated by TNF, TNF receptor (TNFR) 1, TNFR1-associated death domain, TNFR-associated factor 2, NF-kappaB-inducing kinase, IkappaB kinase, and the p65 subunit of NF-kappaB. NF-kappaB-regulated gene products involved in cell survival [inhibitor of apoptosis (IAP) 1, IAP2, X chromosome-linked IAP, Bcl-xL, and survivin], proliferation (cyclin D1 and cyclooxygenase-2), and invasion (matrix metalloproteinase-9) were down-regulated by morin. These effects correlated with enhancement of apoptosis induced by TNF and chemotherapeutic agents., Conclusion: Overall, our results indicate that morin suppresses the activation of NF-kappaB and NF-kappaB-regulated gene expression, leading to enhancement of apoptosis. This may provide the molecular basis for the ability of morin to act as an anticancer and anti-inflammatory agent.