Parthenolide Research Articles

Collateral Sensitivity of Parthenolide via NF-κB and HIF-α Inhibition and Epigenetic Changes in Drug-Resistant Cancer Cell Lines.

Parthenolide (PT) is a sesquiterpene lactone isolated from Tanacetum parthenium. In this study, PT showed varying cytotoxic effects against different solid tumor cell lines. HCT116 (p53+/+) colon carcinoma cells and their parental HCT116 knockout p53 (p53-/-) cell lines showed a resistance degree of 2.36. On the other hand, wild-type U87.MG cells or cells transfected with a deletion-activated EGFR cDNA (U87.MGΔEGFR) exhibited slight sensitivity toward PT. Multidrug-resistant MDA-MB-231-BCRP cells were even more sensitive toward PT than sensitive MDA-MB-231-pcDNA cells with a resistance degree of 0.07 (collateral sensitivity). To the best of our knowledge, hypersensitivity (collateral sensitivity) in MDA-MB-231-BCRP cell line is reported in this study for the first time. We attempted to identify the mechanism of collateral sensitivity. Firstly, we found that PT bound to IKK preventing IκBα degradation and eventually inhibition of the nuclear factor kappa B (NF-κB) pathway. Down-regulation of hypoxia inducing factor 1-alpha (HIF-1α) in MDA-MB-231-BCRP resistant cells may be a second mechanism, since it is a target gene of NF-κB. Moreover, PT also showed epigenetic effect by inhibition of HDAC activity as shown using both molecular docking and HDAC activity assay. Based on COMPARE and hierarchical cluster analyses, we found gene expression profiles that predicted sensitivity or resistance of 47 tumor cell lines toward PT. Interestingly, pathway analyses of gene expression profiles revealed NF-κB and HIF signaling as top networks of these genes, cellular functions and canonical pathways influencing the activity of PT against tumor cells. In conclusion, PT exerted profound cytotoxic activity against various cancer cell lines mainly against BCRP-overexpressing tumor cells, suggesting PT as novel candidate for cancer treatment.

Parthenolide, a feverfew-derived phytochemical, ameliorates obesity and obesity-induced inflammatory responses via the Nrf2/Keap1 pathway

Parthenolide (PL) is one of the most abundant sesquiterpene lactones found in the plant feverfew (Tanacetum parthenium (L.) Sch.Bip.). PL was investigated for its effect on obesity and obesity-induced inflammatory/oxidant responses in vitro and in vivo. An obesity-induced inflammatory response was induced in various co-culture systems using adipocytes (3T3-L1) and macrophages (RAW264.7) in vitro and the effect of PL and its mechanism of action were determined. PL effectively suppressed the adiposity-induced inflammatory responses by downregulating IL-6 (40–42%) and MCP-1 (26–37%) in 3T3-CM-cultured macrophages and contact co-culture system. PL also favorably regulated the dysregulations of adiponectin and resistin in macrophage-conditioned medium (RAW-CM)-cultured adipocytes. In transwell system of adipocyte and macrophage, PL was shown to upregulated Nrf2 and its target molecule, HO-1 by promoting nuclear translocation of Nrf2. In particular, in siRNA knockdown study, the PL-mediated anti-inflammatory response was exerted via the Nrf2/Keap1 pathway. In animal study using high-fat diet (HFD)-fed mice, PL-administered mice showed a significant reduction in body weight and white adipose tissues (WATs). This PL-mediated anti-obese effect was connected to anti-inflammatory responses with the regulation of inflammatory cytokines, and the downregulation of NF-κB and MAPKs. Furthermore, PL differentially modulated CD11c and CD206, which are pro-/anti-inflammatory phenotypes of ATMs, in stroma vascular fraction (SVF) and immunohistochemistry (IHC) staining analyses. PL also regulated the level of (anti)oxidant molecules with the activation of Nrf2/Keap1signaling. Taken together, PL inhibited obesity and obesity-induced inflammatory responses via the activation of Nrf2/Keap1 signaling, indicating a potential of PL as a functional agent to control obesity-related diseases.

Comparison of the inhibition potential of parthenolide and micheliolide on various UDP-glucuronosyltransferase isoforms

Parthenolide (PTL) and micheliolide (MCL) are sesquiterpene lactones with similar structures, and both of them have been reported to exhibit multiple biochemical and pharmacological activities. This study aims to investigate the inhibition of these two compounds on the activity of UDP-glucuronosyltransferases (UGTs).In vitro incubation mixture for recombinant UGTs-catalyzed glucuronidation metabolism of 4-methylumbelliferone (4-MU) was utilized to investigate the inhibition potential. Inhibition kinetics (including inhibition type and parameters) were determined, and in silico docking was employed to elucidate the inhibition difference between PTL and MCL on UGT1A1.MCL showed no inhibition toward all the UGT isoforms, and PTL showed strong inhibition toward UGT1A1. The half-maximal inhibitory concentration (IC50) of PTL on the activity of UGT1A1 was determined to be 64.4 μM. Inhibition kinetics determination showed that PTL exerted noncompetitive inhibition toward UGT1A1, and the inhibition kinetic constant (Ki) was determined to be 12.1 μM. In silico docking method has been employed to show that hydrogen bonds between PTL and the activity cavity of UGT1A1 contributed to the stronger inhibition of PTL on the activity of UGT1A1 than MCL. In conclusion, PTL can more easily induce drug–drug interaction (DDI) with clinical drugs mainly undergoing UGT1A1-catalyzed glucuronidation.

Effects of combined elicitors on parthenolide production and expression of parthenolide synthase (TpPTS) in Tanacetum parthenium hairy root culture

Feverfew (Tanacetum parthenium) is a medicinal herb that has multiple pharmacologic properties. This plant is rich parthenolide (PN). Hairy roots (HRs) culture is a valuable biotechnological tool for the production of plant secondary metabolites. Furthermore, application of elicitors is one of the most effective strategies to induce the metabolite biosynthesis. This study has investigated the effects of various elicitors on the production of PN in feverfew hairy root culture and the expression of parthenolide synthase (TpPTS) gene. Three elicitors were used separately and in combination, including yeast extract (YE) (2.5 mgL− 1), methyl jasmonate (MJ) (100 µM), Ag+ (100 µM), YE + MJ, Ag+ + MJ, YE + Ag+, and YE + MJ + Ag+ which were added to the 1/2MS medium on day 18 and after 48 h and 96 h, hairy roots were collected to determine the PN content. Total RNA was extracted from hairy roots and used for cDNA synthesis and real-time PCR analysis to investigate the gene expression of TpPTS. The PN content was determined by HPLC. The highest PN content (0.05 mg g− 1 dry weight) was achieved after application of elicitors (in 2.5 mgL− 1 YE and 100 µM MJ) and the highest expression of TpPTS gene also occurred after 96 h in the same treatment. It was concluded that some of the combined elicitors could efficiently increase the parthenolide content of feverfew in vitro culture. The additive effect of elicitors might be due to the separate mechanisms of each substance that is acting in an independent pathway.

Pharmacological Inhibition of NF\u03baB Reduces Prostate Cancer Related Osteoclastogenesis In Vitro and Osteolysis Ex Vivo

NFκB is implicated in cancer and bone remodelling, and we have recently reported that the verified NFκB inhibitor Parthenolide (PTN) reduced osteolysis and skeletal tumour growth in models of metastatic breast cancer. Here, we took advantage of in vitro and ex vivo bone cell and organ cultures to study the effects of PTN on the ability of prostate cancer cells and their derived factors to regulate bone cell activity and osteolysis. PTN inhibited the in vitro growth of a panel of human, mouse and rat prostate cancer cells in a concentration-dependent manner with a varying degree of potency. In prostate cancer cell—osteoclast co-cultures, the rat Mat-Ly-Lu, but not human PC3 or mouse RM1-BT, enhanced RANKL stimulated osteoclast formation and PTN reduced these effects without affecting prostate cancer cell viability. In the absence of cancer cells, PTN reduced the support of Mat-Ly-Lu conditioned medium for the adhesion and spreading of osteoclast precursors, and survival of mature osteoclasts. Pre-exposure of osteoblasts to PTN prior to the addition of conditioned medium from Mat-Ly-Lu cells suppressed their ability to support the formation of osteoclasts by inhibition of RANKL/OPG ratio. PTN enhanced the ability of Mat-Ly-Lu derived factors to increase calvarial osteoblast differentiation and growth. Ex vivo, PTN enhanced bone volume in calvaria organ—Mat-Ly-Lu cell co-culture, without affecting Mat-Ly-Lu viability or apoptosis. Mechanistic studies in osteoclasts and osteoblasts confirmed that PTN inhibit NFκB activation related to derived factors from Mat-Ly-Lu cells. Collectively, these findings suggest that pharmacological inhibition of the skeletal NFκB signalling pathway reduces prostate cancer related osteolysis, but further studies in the therapeutic implications of NFκB inhibition in cells of the osteoblastic lineage are needed.

Combining Parthenolide and Sulfasalazine Improves Toxicity to CD200+ LIC Populations in Childhood ALL

CD200 is an immunomodulatory molecule overexpressed in childhood B cell precursor (BCP) acute lymphoblastic leukaemia (ALL). In minimal residual disease (MRD) low risk cases it has been shown that only CD200+ cells could engraft and self-renew in NOD.Cg-PrkdcscidIl2rgtm1Wjl (NSG) mice, making CD200 a very attractive target for these cases. Parthenolide (PTL) has been shown to eradicate CD34+/CD19+, CD34+/CD19- and CD34- leukaemia initiating cell (LIC) populations in BCP ALL. However, the effects of PTL on CD200 subpopulations are not known. In vivo responses to therapy can be restricted by components of the bone marrow microenvironment. Mesenchymal stem cells (MSC) have been shown to protect T-ALL cells from PTL by modulating reactive oxygen species (ROS) activity. This protection could be reversed with the use of sulfasalazine (SSZ), an inhibitor of the cystine uptake transporter xc-. SSZ blocks cysteine generation and release in MSC resulting in elevated ROS levels in T-ALL and increased sensitivity to PTL. This study investigated the effects of PTL and SSZ on unsorted, CD200+ and CD200- BCP-ALL subpopulations in the presence of MSC. Six MRD low risk and 4 risk cases were co-cultured on MSC, at a 2:1 ratio, 1 hour prior to addition of PTL at various doses up to 10mM. After 24 hours, cells were stained with Annexin V and PI and viability was assessed by flow cytometry. In the absence of MSC, low risk cases were slightly less sensitive to PTL with an IC50 of 2.23mM compared to risk ones (IC50 1.54mM, P=0.61). Interestingly, 4 of 6 low risk cases, which were less sensitive to PTL, had normal karyotypes, while the 2 responsive cases had t(12;21). When unsorted ALL cells were co-cultured with MSC, cell survival significantly increased following PTL treatment at doses up to 7.5mM. In low risk cases viability increased from 8.8±19.4% to 30.0±20.4% at 7.5mM PTL (P<0.0001) and in the risk group from 7.2±8.5% to 26.2±20.8% (P=0.04). To investigate whether the protective effect of MSC could be reversed and sensitivity to PTL re-established, SSZ (300mM) was added to MSC cultures 1 hour prior to the addition of ALL cells. In low risk cases, addition of SSZ significantly reduced survival from 47.9±26.2% to 36.0±21.5% at 5mM PTL (P=0.02) and from 30.0±20.4% to 13.7±11.5% at 7.5mM (P=0.001). In risk cases, SSZ reduced the IC50 from 4.29 to 3.37mM PTL and ALL viability decreased from 25±38% to 12±13% at 10mM. PTL was effective against all subpopulations in a subset of 3 low risk and 3 risk cases sorted into CD34+/CD200+, CD34+/CD200-, CD34-/CD200+ and CD34-/CD200- subfractions. In low risk cases, IC50 ranged from 1.3 to 1.8mM in CD34+/CD200+ and CD34-/CD200+ subpopulations with CD34-/CD200- cells slightly less responsive (IC50 2.4mM). Similar results were observed in risk cases, with IC50 ranging from 0.64mM in CD34+/CD200- cells to 3.1mM in CD34-/CD200- cells. The presence of MSC had limited effects on survival of 3 of these subpopulations in low risk cases where viability increased by only 1.5 to 3 fold after PTL treatment. Interestingly, in the CD34+/CD200+ subpopulation, median viability improved 43 fold from 0.7% to 30.2% (range 0.2-34.2%, P=0.32). SSZ reversed this MSC protection and ALL survival following PTL treatment was almost halved (16.9% viable, range 1.6-30.1%, P=0.59). A modest protective effect against PTL was also observed in 3 subpopulations in risk cases with viabilities 1.8 to 2.8 fold higher in the presence of MSC. CD34-/CD200+ cells benefited most from MSC support and median viability increased 21 fold from 3.0% to 63% (range 2.2-90.4%, P=0.5). The addition of SSZ reversed this protective effect and ALL survival was reduced to levels similar to those observed with PTL alone. In conclusion, this study has shown that PTL was very effective against unsorted and all CD200+ or CD200- subpopulations in both low risk and risk BCP ALL cases, confirming its potential as an effective agent in ALL, which is not limited by surface marker expression on target cells. While sensitivity to PTL was reduced in the presence of MSC, it was largely regained with the use of SSZ in the unsorted and CD34+/CD200+ cells in low risk cases and CD34-/CD200+ cells in the risk cases, indicating CD200+ LIC could be effectively targeted using this combination. These data highlight the importance of combining cancer therapies with agents that restrict the effects of the microenvironmental niche to overcome drug resistance in cancer. DisclosuresNo relevant conflicts of interest to declare.

Effects of parthenolide on proliferation inhibiting and cell cycle of human renal cancer 786-O cells

Objective To explore the effects of parthenolide on growth inhibition, cell aoptosis and molecular mechanism of human 786-O kidney cancer cells. Methods The inhibitory effect of parthenolide on human786-O cells was determined by MTT assay.The cell apoptosis and the cell cycle were analyzed by flow cytometry.Bax and the gene change of Bax and Bcl-2 were analyzed by fluorescence quantitative PCR, Bax and Bcl-2 family proteins were analyzed by Westerm blot. Results Parthenolide inhibited the proliferation of 786-O cells in a dose-dependent and time-dependent manner.As showed by flow cytometry, parthenolide treatment of the 786-O cells led to significant G1-phase cell cycle arrest in a dose-dependent manner and a decrease in cell population in the S-phase, whereas the population of cells in G2/M phase did not chang significantly.Flow cytometIy also showed a dose-dependent increase in 786-O cells apoptosis by parthenolide treatment.Westem blot analysis indicated a down-regulation of phosphorylated CyclinDl in a dose-dependent manner in 786-O cells by parthenolide treatment.In the gene and protein level, parthenolide treatment to 786-O cell lines resulted a significant reduction in Bcl-2 expression but an increase in Bax protein in a dose-dependent manner. Conclusions Parthenolide treatment can 1ead to a significant dose-dependent and time-dependent inhibition in the growth and an increase in apoptosis of human 786-O Kidney cancer cells.The mechanism of action of parthenolide may be related to inhibition of CyclinDl activation and regulation of Bcl-2 and Bax expression in human 786-O Kidney cancer cells. Key words: Kidney Neoplasms; Tanacetum parthenium; Cell Proliferation; Apoptosis; Cell Cycle

Parthenolide and DMAPT induce cell death in primitive CML cells through reactive oxygen species.

Tyrosine kinase inhibitors (TKI) have become a first‐line treatment for chronic myeloid leuakemia (CML). TKIs efficiently target bulk CML cells; however, they are unable to eliminate the leukaemic stem cell (LSC) population that causes resistance and relapse in CML patients. In this study, we assessed the effects of parthenolide (PTL) and dimethyl amino parthenolide (DMAPT), two potent inhibitors of LSCs in acute myeloid leukaemia (AML), on CML bulk and CML primitive (CD34+lin−) cells. We found that both agents induced cell death in CML, while having little effect on the equivalent normal hematopoietic cells. PTL and DMAPT caused an increase in reactive oxygen species (ROS) levels and inhibited NF‐κB activation. PTL and DMAPT inhibited cell proliferation and induced cell cycle arrest in G0 and G2 phases. Furthermore, we found cell cycle inhibition to correlate with down‐regulation of cyclin D1 and cyclin A. In summary, our study shows that PTL and DMAPT have a strong inhibitory effect on CML cells. Given that cell cycle arrest was not dependent on ROS induction, we speculate that this effect could be a direct consequence of NF‐κB inhibition and if this mechanism was to be evaded, PTL and DMAPT induced cell death would be potentiated.

Inhibition of NF-\u03baB Reduces Renal Inflammation and Expression of PEPCK in Type 2 Diabetic Mice

Renal gluconeogenesis is markedly promoted in patients with type 2 diabetes mellitus (T2DM); however, the underlying mechanism remains largely unknown. Renal gluconeogenesis is found to be negatively regulated by insulin. T2DM is characterized by chronic and subacute inflammation; however, inflammation has been well recognized to induce insulin resistance. Therefore, this study aimed to investigate whether the enhanced renal gluconeogenesis in T2DM was partially due to the renal inflammation-mediated insulin resistance. If so, whether inflammation inhibitor could partially reverse such change. Diabetic db/db mice and db/m mice were used in our study. Typically, diabetic db/db mice were intraperitoneally treated with 1 mg/kg NF-κB inhibitor parthenolide (PTN) or saline as control every other day. Twelve weeks after treatment, animal samples were collected for measurements. Our results suggested that the expression levels of the inflammatory factors and the gluconeogenic rate-limiting enzyme phosphoenolpyruvate carboxykinase (PEPCK) were up-regulated in renal cortex of both db/db mice and T2DM patients. Moreover, reduced insulin signaling, as well as up-regulated expression of downstream genes FOXO1 and PGC-1ɑ, could be detected in renal cortex of db/db mice compared with that of db/m mice. Consistent with our hypothesis, PTN treatment could alleviate renal inflammation and insulin resistance in db/db mice. Moreover, it could also down-regulate the renal expression of PEPCK, indicating that inflammation could be one of the triggers of insulin resistance and the enhanced renal gluconeogenesis in db/db mice. This study can shed light on the role of inflammation in the enhanced renal gluconeogenesis in T2DM, which may yield a novel target for hyperglycemia.

Crucial role of oxidative stress in bactericidal effect of parthenolide against Xanthomonas oryzae pv. oryzae.

Xanthomonas oryzae pv. oryzae (Xoo) causes rice bacterial blight, which is one of the most devastating diseases on rice. Parthenolide (PTL) is a sesquiterpene lactone possessing multiple bioactivities. In the preliminary study, we found PTL can totally inhibit the growth of Xoo at 10 mg L-1 in vitro. In this study, we aim to further evaluate the anti-bacterial activity of PTL against Xoo and discern the role of oxidative stress in its bactericidal effect. PTL was effective against Xoo both in vitro and in vivo. PTL induced reactive oxygen species (ROS) accumulation in Xoo, leading to cell death, while exogenous catalase can fully abolish its bactericidal effect. PTL sensitivity of catalase deletion mutants of Xoo increased significantly compared with that of wild-type Xoo strain. In addition, PTL treatment increased glutathione peroxidase activity and decreased glutathione (GSH) reductase activity in Xoo, but had no effect on its catalase and superoxide dismutase activities. Interestingly, PTL dramatically reduced the GSH level in Xoo, resulting in disturbed GSH/GSSG balance. Moreover, PTL rapidly reacted with GSH by a nucleophilic addition reaction. PTL is a promising lead compound for developing bactericide against Xoo. PTL rapidly reacts with GSH, resulting in disturbed GSH/GSSG balance in Xoo, which causes ROS accumulation, leading to cell death. Oxidative stress plays a critical role in the bactericidal effect of PTL against Xoo. © 2018 Society of Chemical Industry.

Epoxymicheliolide, a novelguaiane-type sesquiterpene lactone, inhibits NF‑κB/COX‑2 signaling pathways by targeting leucine281 and leucine25 in IKKβ in renal cell carcinoma.

Parthenolide (PTL) is a sesquiterpene lactone compound obtained from Tanacetum parthenium (feverfew) and inhibits the activation of nuclear factor (NF)-κB. Epoxymicheliolide (EMCL) is a compound which is structurally related to PTL; however, EMCL is more stable under acidic and alkaline conditions. As a biologically active molecule, the detailed mechanism by which EMCL inhibits tumor activity remains to be elucidated. The present study evaluated the effect of EMCL on renal cell carcinoma (RCC) cells and identified the underlying mechanisms. It was found that treatment with EMCL significantly inhibited the proliferation of RCC cells in vitro and increased the induction of apoptosis by activating the mitochondria- and caspase-dependent pathway. Simultaneously, EMCL suppressed cell invasion and metastasis by inhibiting epithelial-mesenchymal transition, as observed in a microfluidic chip assay. Furthermore, using immunofluorescence analysis, an electrophoretic mobility shift assay and a dual-luciferase reporter assay, it was shown that treatment with EMCL significantly suppressed the expression of cyclooxygenase-2 by inhibiting the translocation of NF-κB p50/p65 and the activity of NF-κB. Collectively, the results indicated that EMCL suppressed tumor growth by inhibiting the activation of NF-κB and suggested that EMCL may be a novel anticancer agent in the treatment of RCC.

Chronic low dose ethanol induces an aggressive metastatic phenotype in TRAMP mice, which is counteracted by parthenolide.

Despite advances in prostate cancer therapy, dissemination and growth of metastases results in shortened survival. Here we examined the potential anti-cancer effect of the NF-κB inhibitor parthenolide (PTL) and its water soluble analogue dimethylaminoparthenolide (DMAPT) on tumour progression and metastasis in the TRansgenic Adenocarcinoma of the Mouse Prostate (TRAMP) model of prostate cancer. Six-week-old male TRAMP mice received PTL (40mg/kg in 10% ethanol/saline), DMAPT (100mg/kg in sterile water), or vehicle controls by oral gavage thrice weekly until palpable tumour formation. DMAPT treatment slowed normal tumour development in TRAMP mice, extending the time-to-palpable prostate tumour by 20%. PTL did not slow overall tumour development, while the ethanol/saline vehicle used to administer PTL unexpectedly induced an aggressive metastatic tumour phenotype. Chronic ethanol/saline vehicle upregulated expression of NF-κB, MMP2, integrin β1, collagen IV, and laminin, and induced vascular basement membrane degradation in primary prostate tumours, as well as increased metastatic spread to the lung and liver. All of these changes were largely prevented by co-administration with PTL. DMAPT (in water) reduced metastasis to below that of water-control. These data suggest that DMAPT has the potential to be used as a cancer preventive and anti-metastatic therapy for prostate cancer. Although low levels of ethanol consumption have not been shown to strongly correlate with prostate cancer epidemiology, these results would support a potential effect of chronic low dose ethanol on metastasis and the TRAMP model provides a useful system in which to further explore the mechanisms involved.

Parthenolide facilitates apoptosis and reverses drug-resistance of human gastric carcinoma cells by inhibiting the STAT3 signaling pathway.

In the present study, SGC-7901/DDP cells were treated with different concentrations of parthenolide (PN) (2.5–15 µmol/l), cisplatin (DDP) (1.25–15 µg/ml) and PN+DDP. The proliferation inhibition rates were measured using an MTT assay, and the synergies of PN and DDP were analyzed. The effect of PN and DDP on SGC-7901/DDP cell proliferation demonstrated a time- and concentration-dependent association, and a synergy between PN and DDP was identified. DAPI staining and flow cytometry results indicated that 15 µmol/l PN significantly induced SGC-7901/DDP apoptosis and G1 phase arrest compared with the untreated control group. Western blotting analysis results indicated that among the apoptosis-associated proteins, there were dose-dependent increases in the protein expression of apoptosis regulator BAX, cellular tumor antigen p53, cleaved caspase-3 and cleaved capase-9, and decreases in apoptosis regulator Bcl-2 and Bcl-xL protein expression levels. Among the cell cycle-associated proteins, cyclin D1 expression was significantly decreased, cyclin-dependent kinase inhibitor 1 expression was significantly increased, and signal transducer and activator of transcription 3 (STAT3) activation was inhibited. Scratch and Transwell assay results revealed that PN significantly inhibited SGC-7901/DDP cell migration, and invasion. The present study demonstrated that PN induces SGC-7901/DDP apoptosis, inhibits SGC-7901/DDP proliferation, migration and invasion, and enhances the drug sensitivity of the cells to DDP. The underlying mechanisms may be associated with inhibition of the STAT3 signaling pathway and regulation of the downstream apoptotic protein and cyclin expression levels.

Synthesis and biological evaluation of dithiocarbamate esters of parthenolide as potential anti-acute myelogenous leukaemia agents

A series of dithiocarbamate esters of parthenolide (PTL) was designed, synthesised, and evaluated for their anti- acute myelogenous leukaemia (AML) activities. The most promising compound 7l showed greatly improved potency against AML progenitor cell line KG1a with IC50 value of 0.7 μM, and the efficacy was 8.7-folds comparing to that of PTL (IC50 = 6.1 μM). Compound 7l induced apoptosis of total primary human AML cells and leukaemia stem cell (LSCs) of primary AML cells while sparing normal cells. Furthermore, 7l suppressed the colony formation of primary human leukaemia cells. Moreover, compound 12, the salt form of 7l, prolonged the lifespan of mice in two patient-derived xenograft models and had no observable toxicity. The preliminary molecular mechanism study revealed that 7l-mediated apoptosis is associated with mitogen-activated protein kinase signal pathway. On the basis of these investigations, we propose that 12 might be a promising drug candidate for ultimate discovery of anti-LSCs drug.

Parthenolide prevents resistance of MDA-MB231 cells to doxorubicin and mitoxantrone: the role of Nrf2

Triple-negative breast cancer is a group of aggressive cancers with poor prognosis owing to chemoresistance, recurrence and metastasis. New strategies are required that could reduce chemoresistance and increases the effectiveness of chemotherapy. The results presented in this paper, showing that parthenolide (PN) prevents drug resistance in MDA-MB231 cells, represent a contribution to one of these possible strategies. MDA-MB231 cells, the most studied line of TNBC cells, were submitted to selection treatment with mitoxantrone (Mitox) and doxorubicin (DOX). The presence of resistant cells was confirmed through the measurement of the resistance index. Cells submitted to this treatment exhibited a remarkable increment of NF-E2-related factor 2 (Nrf2) level, which was accompanied by upregulation of catalase, MnSOD, HSP70, Bcl-2 and P-glycoprotein. Moreover, as a consequence of overexpression of Nrf2 and correlated proteins, drug-treated cells exhibited a much lower ability than parental cells to generate ROS in response to a suitable stimulation. The addition of PN (2.0 μM) to Mitox and DOX, over the total selection time, prevented both the induction of resistance and the overexpression of Nrf2 and correlated proteins, whereas the cells showed a good ability to generate ROS in response to adequate stimulation. To demonstrate that Nrf2 exerted a crucial role in the induction of resistance, the cells were transiently transfected with a specific small interfering RNA for Nrf2. Similarly to the effects induced by PN, downregulation of Nrf2 was accompanied by reductions in the levels of catalase, MnSOD, HSP70 and Bcl-2, prevention of chemoresistance and increased ability to generate ROS under stimulation. In conclusion, our results show that PN inhibited the development of the resistance toward Mitox and DOX, and suggest that these effects were correlated with the prevention of the overexpression of Nrf2 and its target proteins, which occurred in the cells submitted to drug treatment.

Parthenolide suppresses hypoxia-inducible factor-1α signaling and hypoxia induced epithelial-mesenchymal transition in colorectal cancer.

Activation of hypoxia-inducible factor 1α (HIF‑1α) is frequently observed in solid tumors and it has been associated with various pathophysiological processes, including epithelial‑mesenchymal transition (EMT). Previously, we reported that parthenolide (PT), an inhibitor of nuclear factor-κB (NF-κB), is a promising anticancer agent because it promotes apoptosis of human colorectal cancer (CRC). Here, we investigated a new molecular mechanism by which PT acts on HIF‑1α and hypoxia contributing to EMT by NF‑κB inhibition. Cell viability, DNA binding activity, vascular cell tube formation and cell motility were studied after treatment of PT in hypoxic or normoxic condition. Moreover, effects of PT on hypoxia signaling and hypoxia-induced EMT signaling were investigated. We also examined the inhibitory effect of PT on CRC progression in xenografts. We demonstrated that PT markedly inhibits hypoxia dependent HIF‑1α activity and angiogenesis by preventing NF-κB activation. We also report that PT decreases the level of proteins associated with glucose metabolism, angiogenesis, development and survival that are regulated by HIF‑1α. Furthermore, we verified that PT protects the morphological change from epithelial to mesenchymal state, inhibits matrix metalloproteinase (MMP) enzyme activity and decreases cell motility involved in the -regulation of the hypoxia-induced EMT markers. In addition, PT inhibits growth in CRC xenograft models and regulates NF‑κB, HIF‑1α and EMT specific marker in tissue specimens. Our data demonstrated that PT can inhibit HIF‑1α signaling and hypoxia-induced EMT, suggesting a novel molecular mechanism for HIF‑1α mediated cancer progression and metastasis.

Pharmacological evidence for the bone-autonomous contribution of the NFκB/β-catenin axis to breast cancer related osteolysis

The NFκB signaling pathway is implicated in breast cancer and bone metastasis. However, the bone-autonomous contribution of NFκB to breast cancer-induced osteolysis is poorly understood. Here, we report that pretreatment of osteoblasts with the sesquiterpene lactone Parthenolide (PTN), a verified NFκB inhibitor, prior to exposure to conditioned medium from human and mouse breast cancer cell lines enhanced osteoblast differentiation and reduced osteoblast ability to stimulate osteoclastogenesis. PTN prevented breast cancer-induced osteoclast formation and reduced the ability of breast cancer cells to prolong osteoclast survival and to inhibit osteoclast apoptosis. In vivo, administration of PTN in immuno-competent mice reduced osteolytic bone loss and skeletal tumour growth following injection of the syngeneic 4T1-BT1 cells and reduced local osteolysis caused by conditioned medium from human and mouse osteotropic breast cancer cell lines. Mechanistic studies revealed that NFκB inhibition by PTN in osteoblasts and osteoclasts was accompanied by a significant increase in β-catenin activation and expression. Collectively, these results raise the possibility that combined targeting of NFκB and β-catenin signalling in the tumour microenvironment may be of value in the treatment of breast cancer related osteolysis.

Osteopontin plays a unique role in resistance of CD34 +/CD123 + human leukemia cell lines KG1a to parthenolide

ObjectivesTo determine if parthenolide (PTL) is cytotoxic for leukemia-like KG1a cells and if it involves in certain molecular-mediated resistance, especially osteopontin (OPN). MethodsPTL/daunorubicin (DNR)-treated KG1a cells were examined for viability using MTT and colony-formation assay, and stained for apoptosis using AV/PI. The gene and protein expression were evaluated by qReal-time PCR and Western blotting analysis, respectively. OPN gene was inhibited by OPN siRNA. The cells were stained for various fractions using PE anti-CD34, FITC anti-CD38 and PerCP anti-CD123. ResultsCell viability and proliferation assay exhibited KG1a cells are relatively refractory to used concentrations of PTL. OPN mRNA and protein levels increased in response to PTL. Suppression of OPN with siRNA increased the cytotoxic effects of PTL on KG1a cells. PTL treatment and OPN siRNA suppression in KG1a cells resulted in a decrease of mRNA expression of AKT, mTOR, β-catenin, and Phosphatase and tensin homolog (PTEN). The sub-population cells of CD34+ and CD123+ from KG1a cells are enriched by PTL treatment. ConclusionParthenolide in spite of the reduction in gene expression of AKT, mTOR or beta-catenin, stimulates the OPN expression in KG1a cells. The OPN expression pattern in KG1a cells could be compatible with CD34+/CD123+ subtype enrichment by PTL which in turn implies OPN's unique role in resistance of cell populations characterized by CD34+/CD123+ phenotype.

Parthenolide Promotes Differentiation of Osteoblasts Through the Wnt/β-Catenin Signaling Pathway in Inflammatory Environments.

Periodontitis is a progressive inflammatory disease initiated by bacterial biofilm adhering to the tooth surface. If left untreated, periodontitis may lead to tooth loss and destruction of the alveolar bone. Regaining the lost alveolar bone is a clinical challenge because of the limited differentiation ability of osteoblasts in inflammatory environments. We have previously shown the anti-inflammatory and antiosteoclastogenic activities of parthenolide (PTL) in human periodontal ligament-derived cells by inhibiting nuclear factor kappa B (NF-κB) signaling, indicating its potential for periodontitis treatment. In this study, we further examined whether PTL could stimulate differentiation of osteoblasts from human alveolar bone in inflammatory conditions and investigated the involvement of the Wnt/β-catenin signaling pathway during this process. The results showed that PTL significantly stimulated alkaline phosphatase activity, mineralization nodule formation, and osteogenesis-related gene/protein expression of osteoblasts under the stimulation of tumor necrosis factor-α (TNF-α). In addition, PTL inhibited the NF-κB/p50 pathway and resisted the inhibition of Wnt/β-catenin signaling induced by TNF-α. Our results indicate that the stimulatory effect of PTL on the differentiation of osteoblasts in inflammatory environments may involve the activation of the Wnt/β-catenin signaling pathway, and PTL may be a promising component for bone regeneration in periodontitis treatment.

Anticancer activity profiling of parthenolide analogs generated via P450-mediated chemoenzymatic synthesis

The plant-derived sesquiterpene lactone parthenolide (PTL) was recently found to possess promising anticancer activity but elaboration of this natural product scaffold for optimization of its pharmacological properties has proven challenging via available chemical methods. In this work, P450-catalyzed C–H hydroxylation of positions C9 and C14 in PTL was coupled to carbamoylation chemistry to yield a panel of novel carbamate-based PTL analogs (‘parthenologs’). These compounds, along with a series of other C9- and C14-functionalized parthenologs obtained via O–H acylation, alkylation, and metal-catalyzed carbene insertion, were profiled for their cytotoxicity against a diverse panel of human cancer cell lines. These studies led to the discovery of several parthenologs with significantly improved anticancer activity (2–14-fold) compared to the parent molecule. Most interestingly, two PTL analogs with high cytotoxicity (LC50∼1–3μM) against T cell leukemia (Jurkat), mantle cell lymphoma (JeKo-1), and adenocarcinoma (HeLa) cells as well as a carbamate derivative with potent activity (LC50=0.6μM) against neuroblastoma cells (SK-N-MC) were obtained. In addition, these analyses resulted in the identification of parthenologs featuring both a broad spectrum and tumor cell-specific anticancer activity profile, thus providing valuable probes for the future investigation of biomolecular targets that can affect cell viability across multiple as well as specific types of human cancers. Altogether, these results highlight the potential of P450-mediated chemoenzymatic C–H functionalization toward tuning and improving the anticancer activity of the natural product parthenolide.