Chulwon Kim

β-caryophyllene oxide inhibits constitutive and inducible STAT3 signaling pathway through induction of the SHP-1 protein tyrosine phosphatase

Constitutive activation of STAT3 is frequently observed and closely linked with proliferation, survival, invasion, metastasis and angiogenesis in tumor cells. In the present study, we investigated whether β‐caryophyllene oxide (CPO), a sesquiterpene isolated primarily from the essential oils of medicinal plants such as guava (Psidium guajava), and oregano (Origanum vulgare L.), can mediate its effect through interference with the STAT3 activation pathway in cancer cells. The effect of CPO on STAT3 activation, associated protein kinases and phosphatase, STAT3‐regulated gene products and apoptosis was investigated using both functional proteomics tumor pathway technology platform and different tumor cell lines. We found that CPO suppressed constitutive STAT3 activation in multiple myeloma (MM), breast and prostate cancer cell lines, with a significant dose‐ and time‐dependent effects observed in MM cells. The suppression was mediated through the inhibition of activation of upstream kinases c‐Src and JAK1/2. Also, vanadate treatment reversed CPO‐induced down‐regulation of STAT3, suggesting the involvement of a tyrosine phosphatase. Indeed, we found that CPO induced the expression of tyrosine phosphatase SHP‐1 that correlated with the down‐regulation of constitutive STAT3 activation. Interestingly, deletion of SHP‐1 gene by siRNA abolished the ability of CPO to inhibit STAT3 activation. The inhibition of STAT3 activation by CPO inhibited proliferation, induced apoptosis and abrogated the invasive potential of tumor cells. Our results suggest for the first time that CPO is a novel blocker of STAT3 signaling cascade and thus has an enormous potential for the treatment of various cancers harboring constitutively activated STAT3.

Capillarisin inhibits iNOS, COX-2 expression, and proinflammatory cytokines in LPS-induced RAW 264.7 macrophages via the suppression of ERK, JNK, and NF-κB activation.

The aerial parts of Artemisia capillaris (Compositae) have been used in traditional Korean medicine as a cholagogic, antipyretic, anti-inflammatory, and diuretic purposes. In our previous study, ethanolic extracts of the plant demonstrated a marked anti-inflammatory effect in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells (J. Korean Soc. Appl. Biol. Chem., 2010, 53, 275–282). In the present study, capillarisin (CPS), a flavone, main constituent of A. capillaris, was examined for its anti-inflammatory activity in the cells. We found that CPS highly suppressed LPS-induced nitric oxide (NO) without exerting cytotoxic effects on RAW 264.7 cells. CPS inhibited the expression of LPS-induced inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) protein and their mRNA in a dose-dependent manner. Also, tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-1β, and prostaglandin E2 (PGE2) secretion were decreased by CPS in LPS-stimulated macrophages. As a result, CPS inhibited proinflammatory cytokines, iNOS, and COX-2, which is attributed to the suppression of LPS-induced ERK, JNK, and nuclear factor-κB (NF-κB) activation. Therefore, we demonstrate here that CPS potentially inhibits the biomarkers related to inflammation through the abrogation of ERK, JNK, and NF-κB p65 activation, and it may be a potential therapeutic candidate for the treatment of inflammatory diseases.

Bergamottin, a natural furanocoumarin obtained from grapefruit juice induces chemosensitization and apoptosis through the inhibition of STAT3 signaling pathway in tumor cells

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.

Chrysanthemum indicum L. extract induces apoptosis through suppression of constitutive STAT3 activation in human prostate cancer DU145 cells.

Chrysanthemum indicum L. has been shown to possess antiinflammatory and anticancer activities, but its molecular targets/pathways are not yet fully understood in tumor cells. In the present study, the potential effects of C. indicum on signal transducer and activator of transcription 3 (STAT3) signaling pathway in different tumor cells were examined. The solvent fractions (hexane, CH2Cl2, EtOAc, and BuOH,) were obtained from a crude extract (80% EOH extract) of C. indicum. The methylene chloride fraction of C. indicum (MCI) exhibited strong cytotoxic activity as compared with the other fractions and clearly suppressed constitutive STAT3 activation against both DU145 and U266 cells, but not MDA‐MB‐231 cells. The suppression of constitutive STAT3 activation by MCI is associated with blocking upstream JAK1 and JAK2, but not Src. MCI downregulated the expression of STAT3‐regulated gene products; this is correlated with the accumulation of the cell cycle at sub‐G1 phase, the induction of caspase‐3 activation, and apoptosis. Moreover, the major components of the MCI were bioactive compounds such as sudachitin, hesperetin, chrysoeriol, and acacetin. Sudachitin, chrysoeriol, and acacetin also exerted significantly cytotoxicity, clearly suppressed constitutive STAT3 activation, and induced apoptosis, although hesperetin did not show any significant effect in DU145 cells. Overall, our results demonstrate that MCI could induce apoptosis through inhibition of the JAK1/2 and STAT3 signaling pathways. Copyright

Ginkgolic Acid Inhibits Invasion and Migration and TGF-β-Induced EMT of Lung Cancer Cells Through PI3K/Akt/mTOR Inactivation

Epithelial‐to‐mesenchymal transition (EMT) is a critical cellular phenomenon regulating tumor metastases. In the present study, we investigated whether ginkgolic acid can affect EMT in lung cancer cells and the related underlying mechanism(s) of its actions. We found that ginkgolic acid C15:1 (GA C15:1) inhibited cell proliferation, invasion, and migration in both A549 and H1299 lung cancer cells. GA C15:1 also suppressed the expression of EMT related genes (Fibronectin, Vimentin, N‐cadherin, MMP‐9, MMP‐2, Twist and Snail) and suppressed TGF‐β‐induced EMT as assessed by reduced expression of mesenchymal markers (Fibronectin, Vimentin, N‐cadherin), MMP‐9, MMP‐2, Twist and Snail. However, GA C15:1 did not affect the expression of various epithelial marker proteins (Occludin and E‐cadherin) in both A549 and H1299 cells. TGF‐β‐induced morphologic changes from epithelial to mesenchymal cells and induction of invasion and migration were reversed by GA C15:1. Finally, GA C15:1 not only abrogated basal PI3K/Akt/mTOR signaling cascade, but also reduced TGF‐β‐induced phosphorylation of PI3K/Akt/mTOR pathway in lung cancer cells. Overall, these findings suggest that GA C15:1 suppresses lung cancer invasion and migration through the inhibition of PI3K/Akt/mTOR signaling pathway and provide a source of potential therapeutic compounds to control the metastatic dissemination of tumor cells. J. Cell. Physiol. 232: 346–354, 2017.

Farnesol inhibits tumor growth and enhances the anticancer effects of bortezomib in multiple myeloma xenograft mouse model through the modulation of STAT3 signaling pathway

Aberrant activation of signal transducer and activator of transcription 3 (STAT3) is frequently observed in multiple myeloma (MM) cancer and can upregulate the expression of several genes involved in proliferation, survival, metastasis, and angiogenesis. The effect of farnesol (FOH) on STAT3 activation, associated protein kinases, its regulated gene products, cellular proliferation, and apoptosis was examined. The in vivo effect of FOH on the growth of human MM xenograft tumors alone and in combination with bortezomib (Bor) in athymic nu/nu female mice was also investigated. We found that FOH suppressed both constitutive and inducible STAT3 activation at Tyr705 in MM cells. The suppression of STAT3 was mediated through the inhibition of activation of upstream JAK1, JAK2, and c-Src kinases. Also, treatment with the protein tyrosine phosphatase (PTP) inhibitor, pervanadate treatment reversed the FOH-induced down-regulation of STAT3, possibly indicating the involvement of a PTP. Indeed, we found that FOH treatment induces the increased expression of SHP-2 protein and knockdown of the SHP-2 gene by small interfering RNA suppressed the ability of FOH to inhibit STAT3 activation. FOH inhibited proliferation and significantly potentiated the apoptotic effects of bortezomib (Bor) in U266 cells. When administered intraperitoneally, FOH enhanced Bor-induced growth suppression of human MM xenograft tumors in athymic nu/nu female mice. Our results suggest that FOH is a novel blocker of STAT3 signaling pathway and exerts both anti-proliferative and apoptotic activities in MM in vitro and in vivo.

Emodin inhibits invasion and migration of prostate and lung cancer cells by downregulating the expression of chemokine receptor CXCR4

Emodin (ED), an anthraquinone derivative, has been found to inhibit proliferation, induce apoptosis, suppress angiogenesis, impede metastasis, and enhance chemotherapy. However, the detailed mechanism of ED related to the regulation of CXC chemokine receptor-4 (CXCR4) gene expression that affects cellular migration and invasion in prostate and lung cancer cells are not fully understood. Recent evidence indicates that the CXCR4/CXCL12 axis is involved in promoting invasion and metastasis in tumors. Thus, novel agents that can downregulate CXCR4 expression have therapeutic potential in repressing cancer metastasis. Among ED and its derivatives, it is found that ED downregulated the expression of both CXCR4 and HER2 without affecting cell viability in tumor cells. The suppression of CXCR4 expression by ED was found to correlate with the inhibition of CXCL12-induced migration and invasion of both DU145 and A549 cells. Besides, neither proteasome inhibition nor lysosomal stabilization had any effect on ED-induced decrease in CXCR4 expression. The basic molecular mechanisms unveiled that the downregulation of CXCR4 was at the transcriptional level, as indicated by downregulation of mRNA expression and suppression of NF-κB activation. Overall, our findings suggest that ED is a novel blocker of CXCR4 expression and, thus, has enormous potential as a powerful therapeutic agent for metastatic cancer.

The Butanol Fraction of Guava (Psidium cattleianum Sabine) Leaf Extract Suppresses MMP-2 and MMP-9 Expression and Activity Through the Suppression of the ERK1/2 MAPK Signaling Pathway

The leaf extract of guava (Psidium cattleianum Sabine) has traditionally been used for the treatment of diarrhea and diabetes in East Asia and other countries. Recently, the leaf extract has been employed in the therapy of cancer, bacterial infections, and inflammation in experimental models. However, the exact mechanisms of how guava leaf extract inhibits tumor metastasis and invasion are still unknown. In the present study, we investigated in detail the molecular mechanism(s) responsible for the potential antimetastatic and antiinvasive effects of the butanol fraction of guava leaf extract (GBF). Interestingly, we observed for the first time that GBF suppressed both matrix metalloproteinases (MMP)-9 and MMP-2 expression and activity in part through the downregulation of the ERK1/2 activation in lung cancer cells. Also, importantly, the major components of the GBF were identified as d-glucuronic acid, quercetin 3-glucuronide, loganin, and xanthyletin by LC-ESI-MS/MS. Collectively, our data indicate that the guava leaf could reduce the metastasis of lung cancer cells and therefore suggest that it could be advantageously used to control the metastatic process.

Abrogation of STAT3 signaling cascade by zerumbone inhibits proliferation and induces apoptosis in renal cell carcinoma xenograft mouse model

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.

β-Caryophyllene oxide potentiates TNFα-induced apoptosis and inhibits invasion through down-modulation of NF-κB-regulated gene products

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.