Kanokkarn Phromnoi

Nimbolide Sensitizes Human Colon Cancer Cells to TRAIL through Reactive Oxygen Species- and ERK-dependent Up-regulation of Death Receptors, p53, and Bax

TNF-related apoptosis-inducing ligand (TRAIL) shows promise as a cancer treatment, but acquired tumor resistance to TRAIL is a roadblock. Here we investigated whether nimbolide, a limonoid, could sensitize human colon cancer cells to TRAIL. As indicated by assays that measure esterase activity, sub-G1 fractions, mitochondrial activity, and activation of caspases, nimbolide potentiated the effect of TRAIL. This limonoid also enhanced expression of death receptors (DRs) DR5 and DR4 in cancer cells. Gene silencing of the receptors reduced the effect of limonoid on TRAIL-induced apoptosis. Using pharmacological inhibitors, we found that activation of ERK and p38 MAPK was required for DR up-regulation by nimbolide. Gene silencing of ERK abolished the enhancement of TRAIL-induced apoptosis. Moreover, our studies indicate that the limonoid induced reactive oxygen species production, which was required for ERK activation, up-regulation of DRs, and sensitization to TRAIL; these effects were mimicked by H2O2. In addition, nimbolide down-regulated cell survival proteins, including I-FLICE, cIAP-1, cIAP-2, Bcl-2, Bcl-xL, survivin, and X-linked inhibitor of apoptosis protein, and up-regulated the pro-apoptotic proteins p53 and Bax. Interestingly, p53 and Bax up-regulation by nimbolide was required for sensitization to TRAIL but not for DR up-regulation. Overall, our results indicate that nimbolide can sensitize colon cancer cells to TRAIL-induced apoptosis through three distinct mechanisms: reactive oxygen species- and ERK-mediated up-regulation of DR5 and DR4, down-regulation of cell survival proteins, and up-regulation of p53 and Bax.

Inhibition of MMP-3 activity and invasion of the MDA-MB-231 human invasive breast carcinoma cell line by bioflavonoids

AbstractAim:Stromelysin 1 (matrix metalloproteinase 3; MMP-3) is an enzyme known to be involved in tumor invasion and metastasis. In this study, flavonoids from vegetables and fruits, such as quercetin, kaempferol, genistein, genistin, and daidzein, were tested for their ability to modulate the secretion and activity of MMP-3 in the MDA-MB-231 breast cancer cell line. In addition, we investigated the in vitro effects of flavonoids on MDA-MB-231 cell invasion.Methods:The toxic concentration range of flavonoids was evaluated using the MTT assay. The ability of MDA-MB-231 cells to invade was evaluated using a modified Boyden chamber system. The activity of MMP-3 was determined by casein zymography. The secretion of MMP-3 was evaluated using Western blotting, casein zymography and confirmed by ELISA.Results:Some putative flavonoids, ie, quercetin and kaempferol (flavonols), significantly inhibited the in vitro invasion of MDA-MB-231 cells in a concentration-dependent manner, with IC50 values of 27 and 30 μmol/L, respectively. Quercetin and kaempferol also reduced MMP-3 activity in a dose-dependent manner, with IC50 values in the range of 30 μmol/L and 45 μmol/L, respectively. None of the flavonoids had a significant effect on the secretion of MMP-3.Conclusion:These data show that the flavonols quercetin and kaempferol have higher anti-invasion potency and higher MMP-3 inhibitory activity than isoflavones genistein, genistin and daidzein. In contrast, neither flavonols nor isoflavones have any effect on MMP-3 secretion.

Embelin Suppresses Osteoclastogenesis Induced by Receptor Activator of NF-κB Ligand and Tumor Cells In vitro through Inhibition of the NF-κB Cell Signaling Pathway

Most patients with cancer die not because of the tumor in the primary site, but because it has spread to other sites. Common tumors, such as breast, multiple myeloma, and prostate tumors, frequently metastasize to the bone. It is now well recognized that osteoclasts are responsible for the osteolysis observed in bone metastases of the tumor. Receptor activator of NF-κB ligand (RANKL), a member of the tumor necrosis factor superfamily and an activator of the NF-κB signaling pathway, has emerged as a major mediator of bone loss, commonly associated with cancer and other chronic inflammatory diseases. Embelin (2,5-dihydroxy-3-undecyl-1,4-benzoquinone), derived from the Ayurvedic medicinal plant Embelia ribes, has been shown to bind and inhibit X-linked inhibitor of apoptosis protein and inhibit inflammatory pathways. We investigated whether embelin could inhibit osteoclastogenesis-associated bone loss induced by RANKL and by tumor cells in vitro. We found that embelin suppressed the RANKL-induced differentiation of monocytes into osteoclasts. This benzoquinone also suppressed the osteoclastogenesis induced by multiple myeloma and by breast cancer cells. This effect of embelin correlated with the suppression of NF-κB activation and inhibition of IκBα phosphorylation and IκBα degradation. Inhibition of IκBα phosphorylation was due to the inhibition of IκBα kinase (IKK) activation. Furthermore, by using an inhibitor of the IKKγ or NF-κB essential modulator (NEMO), the regulatory component of the IKK complex, we showed that the NF-κB signaling pathway is mandatory for RAW 264.7 cell differentiation into osteoclasts. Thus, embelin, an inhibitor of RANKL-induced NF-κB activation has great potential as a therapeutic agent for osteoporosis and cancer-linked bone loss. Mol Cancer Res; 8(10); 1425–36. ©2010 AACR.

Thiocolchicoside Exhibits Anticancer Effects through Downregulation of NF-κB Pathway and Its Regulated Gene Products Linked to Inflammation and Cancer

The discovery of new uses for older, clinically approved drugs is one way to expedite drug development for cancer. Thiocolchicoside, a semisynthetic colchicoside from the plant Gloriosa superba, is a muscle relaxant and used to treat rheumatologic and orthopedic disorders because of its analgesic and anti-inflammatory mechanisms. Given that activation of the transcription factor NF-κB plays a major role in inflammation and tumorigenesis, we postulated that thiocolchicoside would inhibit NF-κB and exhibit anticancer effects through the modulation of NF-κB–regulated proteins. We show that thiocolchicoside inhibited proliferation of leukemia, myeloma, squamous cell carcinoma, breast, colon, and kidney cancer cells. Formation of tumor colonies was also suppressed by thiocolchicoside. The colchicoside induced apoptosis, as indicated by caspase-3 and poly(ADP-ribose) polymerase cleavage, and suppressed the expression of cell survival [e.g., Bcl-2, X-linked inhibitor of apoptosis (XIAP), MCL-1, bcl-xL, cIAP-1, cIAP-2, and cFLIP] proteins. Cell proliferation biomarkers such as c-MYC and phosphorylation of phosphoinositide 3-kinase and glycogen synthase kinase 3β were also blocked by thiocolchicoside. Because most cell survival and proliferation gene products are regulated by NF-κB, we studied the effect of thiocolchicoside on this transcription factor and found that thiocolchicoside inhibited NF-κB activation, degradation of inhibitory κBα (IκBα), IκBα ubiquitination, and phosphorylation, abolished the activation of IκBα kinase, and suppressed p65 nuclear translocation. This effect of thiocolchicoside on the NF-κB pathway led to inhibition of NF-κB reporter activity and cyclooxygenase-2 promoter activity. Our results indicate that thiocolchicoside exhibits anticancer activity through inhibition of NF-κB and NF-κB–regulated gene products, which provides novel insight into a half-century old drug. Cancer Prev Res; 3(11); 1462–72. ©2010 AACR.

Dihydroxypentamethoxyflavone Down-Regulates Constitutive and Inducible Signal Transducers and Activators of Transcription-3 through the Induction of Tyrosine Phosphatase SHP-1

Because constitutive activation of signal transducers and activators of transcription-3 (STAT3) has been linked with cellular transformation, survival, proliferation, chemoresistance, and angiogenesis of various tumor cells, agents that can suppress STAT3 activation have potential as cancer therapeutics. In the present report, we identified a flavone from the leaves of a Thai plant, Gardenia obtusifolia, 5,3′-dihydroxy-3,6,7,8,4′-pentamethoxyflavone (PMF), that has the ability to inhibit STAT3 activation. PMF inhibited both constitutive and interleukin-6-inducible STAT3 activation in multiple myeloma (MM) cells, as indicated by suppression of STAT3 phosphorylation, nuclear translocation, DNA binding, and STAT3-regulated gene expression. The inhibition of STAT3 by PMF was reversible. We found that the activation of various kinases including Janus-like kinase (JAK)-1, JAK-2, c-Src, extracellular signal-regulated kinases 1 and 2, AKT, and epidermal growth factor receptor, implicated in STAT3 activation, were inhibited by the flavone. It is noteworthy that pervanadate suppressed the ability of PMF to inhibit the phosphorylation of STAT3, suggesting that protein tyrosine phosphatase was involved. PMF induced the expression of SHP-1 and was linked to the dephosphorylation of STAT3, because its deletion by small interfering RNA abolished the PMF-induced constitutive and inducible STAT3 inhibition. STAT3 inhibition led to the suppression of proteins involved in proliferation (cyclin D1 and c-myc), survival (survivin, Mcl-1, Bcl-xL, Bcl-2, and cIAP-2), and angiogenesis (vascular endothelial growth factor). Finally, PMF inhibited proliferation and induced apoptosis of MM cells. PMF also significantly potentiated the apoptotic effects of Velcade and thalidomide in MM cells. Overall, these results suggest that PMF is a novel blocker of STAT3 activation and thus may have potential in suppression of tumor cell proliferation and reversal of chemoresistance in MM cells.

Thiocolchicoside suppresses osteoclastogenesis induced by RANKL and cancer cells through inhibition of inflammatory pathways: a new use for an old drug

BACKGROUND AND PURPOSE Most patients with cancer die not because of the tumour in the primary site, but because it has spread to other sites. Common tumours, such as breast, multiple myeloma, and prostate tumours, frequently metastasize to the bone. To search for an inhibitor of cancer‐induced bone loss, we investigated the effect of thiocolchicoside, a semi‐synthetic colchicoside derived from the plant Gloriosa superba and clinically used as a muscle relaxant, on osteoclastogenesis induced by receptor activator of NF‐κB ligand (RANKL) and tumour cells.

A novel pentamethoxyflavone down-regulates tumor cell survival and proliferative and angiogenic gene products through inhibition of IκB kinase activation and sensitizes tumor cells to apoptosis by cytokines and chemotherapeutic agents.

Most anticancer drugs have their origin in traditional medicinal plants. We describe here a flavone, 5,3′-dihydroxy-3,6,7,8,4′-pentamethoxyflavone (PMF), from the leaves of the Thai plant Gardenia obtusifolia, that has anti-inflammatory and anticancer potential. Because the nuclear factor-κB (NF-κB) pathway is linked to inflammation and tumorigenesis, we investigated the effect of PMF on this pathway. We found that PMF suppressed NF-κB activation induced by inflammatory agents, tumor promoters, and carcinogens. This suppression was not specific to the cell type. Although PMF did not directly modify the ability of NF-κB proteins to bind to DNA, it inhibited IκBα (inhibitory subunit of NF-κB) kinase, leading to suppression of phosphorylation and degradation of IκBα, and suppressed consequent p65 nuclear translocation, thus abrogating NF-κB-dependent reporter gene expression. Suppression of the NF-κB cell signaling pathway by the flavone led to the inhibition of expression of NF-κB-regulated gene products that mediate inflammation (cyclooxygenase-2), survival (XIAP, survivin, Bcl-xL, and cFLIP), proliferation (cyclin D1), invasion (matrix metalloproteinase-9), and angiogenesis (vascular endothelial growth factor). Suppression of antiapoptotic gene products by PMF correlated with the enhancement of apoptosis induced by tumor necrosis factor-α and the chemotherapeutic agents cisplatin, paclitaxel, and 5-flurouracil. Overall, our results indicate that PMF suppresses the activation of NF-κB and NF-κB-regulated gene expression, leading to the enhancement of apoptosis. This is the first report to demonstrate that this novel flavone has anti-inflammatory and anticancer effects by targeting the IKK complex.

Erratum: A novel pentamethoxyflavone down-regulates tumor cell survival and proliferative and angiogenic gene products through inhibition of IκB kinase activation and sensitizes tumor cells to apoptosis by cytokines and chemotherapeutic agents (Molecular Pharmacology (2010) 79 (279-289))

Most anticancer drugs have their origin in traditional medicinal plants. We describe here a flavone, 5,3′-dihydroxy-3,6,7,8,4′-pentamethoxyflavone (PMF), from the leaves of the Thai plant Gardenia obtusifolia, that has anti-inflammatory and anticancer potential. Because the nuclear factor-κB (NF-κB) pathway is linked to inflammation and tumorigenesis, we investigated the effect of PMF on this pathway. We found that PMF suppressed NF-κB activation induced by inflammatory agents, tumor promoters, and carcinogens. This suppression was not specific to the cell type. Although PMF did not directly modify the ability of NF-κB proteins to bind to DNA, it inhibited IκBα (inhibitory subunit of NF-κB) kinase, leading to suppression of phosphorylation and degradation of IκBα, and suppressed consequent p65 nuclear translocation, thus abrogating NF-κB-dependent reporter gene expression. Suppression of the NF-κB cell signaling pathway by the flavone led to the inhibition of expression of NF-κB-regulated gene products that mediate inflammation (cyclooxygenase-2), survival (XIAP, survivin, Bcl-xL, and cFLIP), proliferation (cyclin D1), invasion (matrix metalloproteinase-9), and angiogenesis (vascular endothelial growth factor). Suppression of antiapoptotic gene products by PMF correlated with the enhancement of apoptosis induced by tumor necrosis factor-α and the chemotherapeutic agents cisplatin, paclitaxel, and 5-flurouracil. Overall, our results indicate that PMF suppresses the activation of NF-κB and NF-κB-regulated gene expression, leading to the enhancement of apoptosis. This is the first report to demonstrate that this novel flavone has anti-inflammatory and anticancer effects by targeting the IKK complex.

Correction: Nimbolide sensitizes human colon cancer cells to TRAIL through reactive oxygen species- and ERK-dependent up-regulation of death receptors, p53, and Bax (Journal of Biological Chemistry (2011) 286 (1134-1146))

Subash C. Gupta, Simone Reuter, Kanokkarn Phromnoi, Byoungduck Park, Padmanabhan S. Hema, Mangalam Nair, and Bharat B. Aggarwal This article has been retracted by the publisher. An investigation at MD Anderson determined that the image of medium control and the image of DR4 DR5 siRNA treated with TRAIL in Fig. 3B were reused. THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 291, NO. 32, p. 16925, August 5, 2016

A Novel Pentamethoxyflavone Downregulates Tumor Cell Survival, Proliferative, and Angiogenic Gene Products Through Inhibition of Activation of IκB Kinase, and Sensitizes Tumor Cells to Apoptosis by Cytokines and Chemotherapeutic Agents

Most anticancer drugs have their origin in traditional medicinal plants. We describe here a flavone, 5,3′-dihydroxy-3,6,7,8,4′-pentamethoxyflavone (PMF), from the leaves of the Thai plant Gardenia obtusifolia, that has anti-inflammatory and anticancer potential. Because the nuclear factor-κB (NF-κB) pathway is linked to inflammation and tumorigenesis, we investigated the effect of PMF on this pathway. We found that PMF suppressed NF-κB activation induced by inflammatory agents, tumor promoters, and carcinogens. This suppression was not specific to the cell type. Although PMF did not directly modify the ability of NF-κB proteins to bind to DNA, it inhibited IκBα (inhibitory subunit of NF-κB) kinase, leading to suppression of phosphorylation and degradation of IκBα, and suppressed consequent p65 nuclear translocation, thus abrogating NF-κB-dependent reporter gene expression. Suppression of the NF-κB cell signaling pathway by the flavone led to the inhibition of expression of NF-κB-regulated gene products that mediate inflammation (cyclooxygenase-2), survival (XIAP, survivin, Bcl-xL, and cFLIP), proliferation (cyclin D1), invasion (matrix metalloproteinase-9), and angiogenesis (vascular endothelial growth factor). Suppression of antiapoptotic gene products by PMF correlated with the enhancement of apoptosis induced by tumor necrosis factor-α and the chemotherapeutic agents cisplatin, paclitaxel, and 5-flurouracil. Overall, our results indicate that PMF suppresses the activation of NF-κB and NF-κB-regulated gene expression, leading to the enhancement of apoptosis. This is the first report to demonstrate that this novel flavone has anti-inflammatory and anticancer effects by targeting the IKK complex.