2'-Hydroxy-4-methylsulfonylchalcone enhances TRAIL-induced apoptosis in prostate cancer cells.

Prostate cancer is the most common malignant cancer in men and the second leading cause of cancer deaths. Previously, we have shown that 2'-hydroxy-4-methylsulfonylchalcone (RG003) induced apoptosis in prostate cancer cell lines PC-3 and DU145. Although tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anticancer agent, some cancer cells are resistant to TRAIL treatment. PC-3 and LNCaP prostatic cancer cell lines have been reported to be resistant to TRAIL-induced apoptosis. Here, we show for the first time that RG003 overcomes TRAIL resistance in prostate cancer cells. RG003 can enhance TRAIL-induced apoptosis through DR5 upregulation and downregulation of Bcl-2, PI3K/Akt, NF-κB, and cyclooxygenase-2 (COX-2) survival pathways. When used in combined treatment, RG003 and TRAIL amplified TRAIL-induced activation of apoptosis effectors and particularly activation of caspase-8 and the executioner caspase-3, leading to increased poly-ADP-ribose polymerase cleavage and DNA fragmentation in prostate cancer cells. Furthermore, we showed that RG003 reduced COX-2 expression in cells. Previously, we showed that COX-2 was involved in resistance to an apoptosis mechanism; then, its inhibition by RG003 could render cells more sensitive to TRAIL treatment. We showed that nuclear factor-κB activation was inhibited after RG003 treatment. This inhibition was correlated with reduction in COX-2 expression and induction of apoptosis. Overall, we conclude, for the first time, that RG003 can enhance TRAIL-induced apoptosis in human prostate cancer cells. The significance of our in-vitro study with RG003 and TRAIL combined is very encouraging, suggesting the relevance of testing this combined treatment in xenograft animal models.

Sonic Hedgehog activation is implicated in diosgenin-induced megakaryocytic differentiation of human erythroleukemia cells.

Differentiation therapy is a means to treat cancer and is induced by different agents with low toxicity and more specificity than traditional ones. Diosgenin, a plant steroid, is able to induce megakaryocytic differentiation or apoptosis in human HEL erythroleukemia cells in a dose-dependent manner. However, the exact mechanism by which diosgenin induces megakaryocytic differentiation has not been elucidated. In this study, we studied the involvement of Sonic Hedgehog in megakaryocytic differentiation induced by diosgenin in HEL cells. First, we showed that different elements of the Hedgehog pathway are expressed in our model by qRT-PCR. Then, we focused our interest on key elements in the Sonic Hedgehog pathway: Smoothened receptor, GLI transcription factor and the ligand Sonic Hedgehog. We showed that Smoothened and Sonic Hedgehog were overexpressed in disogenin-treated cells and that GLI transcription factors were activated. Then, we showed that SMO inhibition using siSMO or the GLI antagonist GANT-61, blocked megakaryocytic differentiation induced by diosgenin in HEL cells. Furthermore, we demonstrated that Sonic Hedgehog pathway inhibition led to inhibition of ERK1/2 activation, a major physiological pathway involved in megakaryocytic differentiation. In conclusion, our study reports, for the first time, a crucial role for the Sonic Hedgehog pathway in diosgenin-induced megakaryocytic differentiation in HEL cells.

Novel methylsulfonyl chalcones as potential antiproliferative drugs for human prostate cancer: involvement of the intrinsic pathway of apoptosis.

Limited success has been achieved in extending the survival of patients with metastatic and hormone-refractory prostate cancer (HRPC). There is a strong need for novel agents in the treatment and prevention of HRPC. In the present study, the apoptotic mechanism of action of RG003 (2'-hydroxy-4-methylsulfonylchalcone) and RG005 (4'-chloro-2'-hydroxy-4-methylsulfonylchalcone) in association with intracellular signalling pathways was investigated in the hormone-independent prostate carcinoma cells PC-3 and DU145. We showed that these compounds induced apoptosis through the intrinsic pathway but not through the extrinsic one. We showed that synthetic chalcones induced an activation of caspase-9 but not caspase-8 in PC-3 cells. Even if both chalcones induced apoptosis in PC-3 cells, a dominant effect of RG003 treatment was observed resulting in a disruption of ∆ψm, caspase-9 and caspase-3 activation, PARP cleavage and DNA fragmentation. Furthermore, in regard to our results, it is clear that the simultaneous inhibition of Akt and NF-κB signalling can significantly contribute to the anticancer effects of RG003 and RG005 in PC-3 prostate cancer cells. NF-κB inhibition was correlated with the reduction of COX-2 expression and induction of apoptosis. Our results clearly indicate for the first time that RG003 and RG005 exert their potent anti­proliferative and pro-apoptotic effects through the modulation of Akt/NF-κB/COX-2 signal transduction pathways in PC-3 prostate cancer cells with a dominant effect for RG003.

Cyclooxygenase-2 positively regulates Akt signalling and enhances survival of erythroleukemia cells exposed to anticancer agents.

Cyclooxygenase-2 (COX-2) has been found to be highly expressed in many types of cancers and to contribute to tumorigenesis via the inhibition of apoptosis, increased angiogenesis and invasiveness. In hematological malignancies, COX-2 expression was found to correlate with poor patient prognosis. However, the exact role of COX-2 expression in these malignancies, and particularly in erythroleukemias, remains unclear. The aim of this work was to describe and understand the relationships between COX-2 expression and apoptosis rate in erythroleukemia cells after apoptosis induction by several anticancer agents. We used three different erythroleukemia cell lines in which COX-2 expression was modulated by transfection with either COX-2 siRNA or COX-2 cDNA. These cellular models were then treated with apoptosis inducers and apoptosis onset and intensity was followed. Cell signalling was evaluated in unstimulated transfected cells or after apoptosis induction. We found that COX-2 inhibition rendered erythroleukemia cells more sensitive to apoptosis induction and that in cells overexpressing COX-2 apoptosis induction was reduced. We demonstrated that COX-2 inhibition decreased the pro-survival Akt signalling and activated the negative regulator of Akt signalling, phosphatase and tensin homologue deleted on chromosome 10 (PTEN). Conversely, in COX-2 overexpressing cells, Akt signalling was activated and PTEN was inhibited. In these last cells, inhibition of casein kinase 2 or Akt signalling restored sensitivity to apoptotic agents. Our findings highlighted that COX-2 can positively regulate Akt signalling mostly through PTEN inhibition, partly via casein kinase 2 activation, and enhances survival of erythroleukemia cells exposed to anticancer agents.

Cyclopamine and jervine induce COX-2 overexpression in human erythroleukemia cells but only cyclopamine has a pro-apoptotic effect.

Erythroleukemia is generally associated with a very poor response and survival to current available therapeutic agents. Cyclooxygenase-2 (COX-2) has been described to play a crucial role in the proliferation and differentiation of leukemia cells, this enzyme seems to play an important role in chemoresistance in different cancer types. Previously, we demonstrated that diosgenin, a plant steroid, induced apoptosis in HEL cells with concomitant COX-2 overexpression. In this study, we investigated the antiproliferative and apoptotic effects of cyclopamine and jervine, two steroidal alkaloids with similar structures, on HEL and TF1a human erythroleukemia cell lines and, for the first time, their effect on COX-2 expression. Cyclopamine, but not jervine, inhibited cell proliferation and induced apoptosis in these cells. Both compounds induced COX-2 overexpression which was responsible for apoptosis resistance. In jervine-treated cells, COX-2 overexpression was NF-κB dependent. Inhibition of NF-κB reduced COX-2 overexpression and induced apoptosis. In addition, cyclopamine induced apoptosis and COX-2 overexpression via PKC activation. Inhibition of the PKC pathway reduced both apoptosis and COX-2 overexpression in both cell lines. Furthermore, we demonstrated that the p38/COX-2 pathway was involved in resistance to cyclopamine-induced apoptosis since p38 inhibition reduced COX-2 overexpression and increased apoptosis in both cell lines.

The P2Y2/Src/p38/COX-2 pathway is involved in the resistance to ursolic acid-induced apoptosis in colorectal and prostate cancer cells.

One of the hallmarks of cancer is resistance to apoptosis. Elucidating the mechanisms of how cancer cells evade or delay apoptosis should lead to novel therapeutic strategies. Previously, we showed that HT-29 colorectal cancer cells undergoing apoptosis overexpressed cyclooxygenase-2 (COX-2), in a p38 dependent pathway, to delay ursolic acid-induced apoptosis. Here, we focused on elucidating the upstream signaling pathways regulating this resistance mechanism. The role of ATP as an extracellular signaling molecule took a long time to be accepted. In recent years, ATP and its analogs, via the activation of specific purinergic receptors, have been implicated in many biological processes including cell proliferation, differentiation and apoptosis. In the present report, we have demonstrated a novel role involving purinergic receptors and particularly the P2Y(2) receptor in resistance to ursolic acid-induced apoptosis in both colorectal HT-29 and prostate DU145 cancer cells. We found that ursolic acid induced an increase in intracellular ATP and P2Y(2) transcript levels. Upon activation, P2Y(2) activated Src which in turn phosphorylated p38 leading to COX-2 overexpression which induced resistance to apoptosis in both HT-29 and DU145 cells. Furthermore, Ca(2+)-independent PLA(2) (iPLA(2)) and Ca(2+)-dependent secretory PLA(2) (sPLA(2)) were responsible for arachidonic acid release, the substrate of COX-2. Our findings document that apoptosis triggering was dependent on protein kinase C (PKC) activation in both cell lines after ursolic acid treatment.

HT-29 colorectal cancer cells undergoing apoptosis overexpress COX-2 to delay ursolic acid-induced cell death.

Colorectal cancer is one of the most common cancer types and the third leading cause of cancer-related death in the western world. Generally, colorectal cancers are resistant to anticancer drugs. Several lines of evidence support a critical role for cyclooxygenase-2 (COX-2) during colorectal tumorigenesis and its role in chemoresistance. In this study, we focused our interest on the role played by COX-2 in apoptosis induced in HT-29 human colorectal cancer cells by ursolic acid (UA), a triterpenoid found in a large variety of plants. We showed that UA-induced apoptosis and that COX-2 was overexpressed only in apoptotic cells. We demonstrated that this overexpression was mediated by the p38 MAP kinase pathway as inhibiting its activation using a p38-specific inhibitor, SB 203580, abrogated COX-2 expression. Inhibiting COX-2 expression either by using a p38-specific inhibitor or COX-2-specific siRNA increased apoptosis. These results demonstrated that COX-2 was involved in a resistance mechanism to UA-induced apoptosis in HT-29 cells. Cells undergoing apoptosis were able to trigger a resistance mechanism by overexpressing a protein such as COX-2 to delay their death. Furthermore, we demonstrated that this resistance mechanism was independent of PGE(2) production as the addition of the specific COX-2 activity inhibitor, NS-398, did not affect apoptosis in UA-treated cells.

Investigating the relationship between cell cycle stage and diosgenin-induced megakaryocytic differentiation of HEL cells using sedimentation field-flow fractionation.

Differentiation therapy could be one strategy for stopping cancer cell proliferation. A plant steroid, diosgenin, is known to induce megakaryocytic differentiation in human erythroleukemia (HEL) cells. In recent studies, the use of sedimentation field-flow fractionation (SdFFF) allowed the preparation of subpopulations that may differ in regard to sensitivity to differentiation induction. The specific goal of this study was to determine the relationship between cell cycle stage and sensitivity to megakaryocytic differentiation induction of HEL cells. After first confirming the capacity of diosgenin to specifically select targets, hyperlayer SdFFF cell sorting was used to prepare fractions according to cell cycle position from crude HEL cells. The sensitivities of these fractions to diosgenin-induced differentiation were then tested. The coupling of SdFFF cell separation to imaging flow cytometry showed that G1-phase cells were more sensitive to differentiation induction than S/G2M-phase cells, confirming the relationship between cell status at the start of induction, the extent of the biological event, and the potential of SdFFF in cancer research.

Antiproliferative effect of extracts from Aristolochia baetica and Origanum compactum on human breast cancer cell line MCF-7.

Aristolochia baetica L. (Aristolochiaceae) and Origanum compactum Benth. (Lamiaceae) are native plants of Morocco used in traditional medicine. In order to systematically evaluate their potential activity on human breast cancer, four different polarity extracts from each plant were assessed in vitro for their antiproliferative effect on MCF-7 cells. As a result, several extracts of those plants showed potent cell proliferation inhibition on MCF-7 cells. Chloroform extract of A. baetica (IC50: 216.06 +/- 15 microg/mL) and ethyl acetate of O. compactum (IC50: 279.51 +/- 16 microg/mL) were the most active. Thin layer chromatography examination of the bioactive extracts of A. baetica and O. compactum showed the presence of aristolochic acid and betulinic acid, respectively. These results call for further studies of these extracts.

Cyclooxygenase-2 and 5-lipoxygenase pathways in diosgenin-induced apoptosis in HT-29 and HCT-116 colon cancer cells.

We studied the relationship between diosgenin-induced apoptosis and arachidonic acid metabolism in two cancer cell lines, the HT-29 and HCT-116. Diosgenin is a steroidal saponin known for its antiproliferative and pro-apoptotic effects on cancer cells. We focused our attention on two enzymes intervening in two different pathways of arachidonic metabolism: cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LOX). HT-29 and HCT-116 cells express 5-LOX but only HT-29 cells express COX-2 since HCT-116 are deficient. After 40 microM diosgenin treatment, we observed apoptosis hallmarks in both cell lines but HT-29 cells were more resistant with delayed apoptosis. In these cells, COX-2 expression and activity were increased and for the first time we showed that diosgenin also increased 5-LOX expression and enhanced leukotriene B4 production. Inhibition of 5-LOX by AA-861 significantly reduced apoptosis in both cell lines but COX-2 inhibition by NS-398 strongly sensitized HT-29 cells to diosgenin-induced apoptosis compared to HCT-116 cells. In this study, we showed the implication of COX-2 and 5-LOX in diosgenin-induced apoptosis but these results demonstrate how difficult it is to assess the correlation between the apoptotic signalling pathway of diosgenin and arachidonic acid metabolism with certitude.

Citral inhibits cell proliferation and induces apoptosis and cell cycle arrest in MCF-7 cells.

Many natural components of plants extract are studied for their beneficial effects on health and particularly on carcinogenesis chemoprevention. In this study, we investigated the effect of citral (3,7-dimethyl-2,6-octadienal), a key component of essential oils extracted from several herbal plants, on the proliferation rate, cell cycle distribution, and apoptosis of the human breast cancer cell line MCF-7. The effects of this compound were also tested on cyclo-oxygenase activity. Citral treatment caused inhibition of MCF-7 cell growth (IC(50)-48 h: 18 x 10(-5)m), with a cycle arrest in G(2)/M phase and apoptosis induction. Moreover, we observed a decrease in prostaglandin E(2) synthesis 48 h after citral treatment. These findings suggest that citral has a potential chemopreventive effect.

Inhibition of human rheumatoid arthritis synovial cell survival by hecogenin and tigogenin is associated with increased apoptosis, p38 mitogen-activated protein kinase activity and upregulation of cyclooxygenase-2.

We conducted our study to assess the antiproliferative and proapoptotic potential of hecogenin and tigogenin, two saponins which are structurally similar to diosgenin. We particularly focused our attention on mitogen-activated protein kinase (MAPK) activation in relation to apoptosis but also with the COX-2 expression and activity. Rheumatoid arthritis (RA) synoviocytes were isolated from fresh synovial biopsies obtained from five RA patients undergoing hip arthroplasty. Measurement of cell proliferation was determined using the MTT assay. Apoptosis was evaluated by studying caspase-8, caspase-9 and caspase-3 activities but also by quantification of DNA fragmentation. Quantification of human phospho-MAPKs was realized by ELISA. COX-2 expression was demonstrated by Western blot analysis and COX-2 activity by assay of endogenous prostaglandin E2 (PGE2) production. Tigogenin was more effective than hecogenin in inducing apoptosis in human RA fibroblast-like synoviocytes (FLS) which was caspase dependent but poly(ADP-ribose) polymerase independent and characterized by DNA fragmentation. Our results demonstrated hecogenin- and tigogenin-induced apoptosis through activation of p38 without affecting the JNK and ERK pathways. Indeed, pretreatment with a p38 inhibitor decreased saponin-induced apoptosis with a significant decrease in DNA fragmentation. Furthermore, the rate of apoptosis induced by hecogenin or tigogenin was associated with overexpression of COX-2 correlated with overproduction of endogenous PGE2. These new results provide strong evidence that a family of structurally similar plant steroids is capable of inducing apoptosis in human RA FLS with different rates and different signalling pathways. This study also confirms the discussed appearance of the downregulation or upregulation of COX-2 in cell apoptosis as a function of cell type.

MAP kinase subtypes and Akt regulate diosgenin-induced apoptosis of rheumatoid synovial cells in association with COX-2 expression and prostanoid production.

In the present study, we investigated the signalling pathways involved in diosgenin-induced apoptosis in human rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) in vitro with particular interest on Akt and MAPKs activation in relation to arachidonic acid metabolism via COX-2 pathway. MAPK activation was measured by ELISA quantification in diosgenin-treated human RA FLS. Expression of Akt and phospho-Akt was analyzed by Western blot analysis. Nuclear factor-kappaB (NF-kappaB) translocation was evaluated by electromobility shift assay. The prostanoid production (COX-2 activity) was measured by quantitative ELISA. Diosgenin-induced apoptosis in the presence of MAPK or Akt inhibitors was detected by a quantitative determination of DNA fragmentation. Treatment of human RA FLS with 40 microM diosgenin caused an activation of p38 and JNK and an inhibition of ERK phosphorylation. Akt and NF-kappaB are potentially required for diosgenin-induced apoptosis in human RA FLS because 40 microM diosgenin abrogated Akt phosphorylation which correlated with an inhibition of NF-kappaB nuclear translocation. SB203580 and SP600125 (p38 and JNK inhibitors) reduced diosgenin-induced DNA fragmentation whereas U0126 and LY294002 (MEK and PI3 kinase/Akt inhibitors) caused an amplification of proapoptotic effect of diosgenin. Diosgenin increased COX-2 activity resulting in PGE2 and 6-keto-PGF1alpha overproduction in human RA FLS. All MAPK inhibitors markedly reduced diosgenin-induced PGE2 and 6-keto-PGF1alpha synthesis except for SP600125 on 6-keto-PGF1alpha production. These results provide, for the first time, strong evidence that a combined association implicating a MEK inhibitor (U0126) and diosgenin is the most effective in inducing very strong apoptosis with down-regulation of COX-2 expression and activity in human RA FLS.

Role of MAPKs and NF-kappaB in diosgenin-induced megakaryocytic differentiation and subsequent apoptosis in HEL cells.

Megakaryocytopoiesis is characterized by progressive polyploidization and acquisition of megakaryo-cytic markers. MAPK pathways play a key role during megakaryocytic differentiation of megakaryocyte precursors or leukemic cells. Apoptosis is the physiological fate of normal megakaryocyte after differentiation and maturation. The aim of this study was to investigate the signaling pathways involved in diosgenin-induced differentiation and the fate of diosgenin-differentiated HEL cells. The present report shows that diosgenin induced megakaryocytic differentiation of HEL cells through a combined activation of ERK and inhibition of the p38 MAPK pathways. Inhibition of ERK activation by a MEK inhibitor abrogated diosgenin-induced differentiation. Afterwards, differentiated cells showed a marked inhibition of expression of survival factors NF-kappaB, Akt and Bcl-xL and activation of caspase-3 together with PARP cleavage leading to apoptotic death of diosgenin-differentiated cells.

Diosgenin, a plant steroid, induces apoptosis in COX-2 deficient K562 cells with activation of the p38 MAP kinase signalling and inhibition of NF-kappaB binding.

Diosgenin is a steroidal sapogenin with antitumor properties. We previously showed that diosgenin induced apoptosis in human erythroleukemia (HEL) cells. In order to elucidate the mechanism of its apoptotic activity, we investigated the effect of diosgenin on nuclear factor-kappaB (NF-kappaB) binding and on three groups of human mitogen-activated protein kinases (MAPKs) in relation to diosgenin-induced apoptosis in different erythroleukemia cell lines (K562 and HEL). Our results showed that diosgenin decreased DNA binding of NF-kappaB in K562 and HEL cells after 48-h diosgenin treatment. This inhibition of NF-kappaB binding was correlated with strong apoptosis in both erythroleukemia cell lines. Diosgenin-induced apoptosis was associated with cyclo-oxygenase-2 (COX-2) up-regulation in HEL cells but not in K562 cells which are COX-2 deficient. Furthermore, diosgenin inhibited extracellular signal-regulated kinase (ERK) activation only in HEL cells. However, diosgenin activated p38 MAPK in both cell lines and activated c-jun NH2-terminal kinases (JNKs) only in HEL cells. Pre-treatment with a selective p38 inhibitor inhibited diosgenin-induced DNA fragmentation in K562 cells. For the first time, our results suggest that inhibition of NF-kappaB nuclear binding and p38 MAPK activation are involved in the diosgenin-mediated signal cascades in K562 cells for inducing/regulating DNA fragmentation.

Effects of the active metabolite of leflunomide, A77 1726, on cytokine release and the MAPK signalling pathway in human rheumatoid arthritis synoviocytes.

Inflammatory cytokines or soluble factors are essential in the pathogenesis of rheumatoid arthritis (RA). Leflunomide is an effective disease modifying antirheumatic drug (DMARD) in RA. The objective of the present study was to evaluate for the first time the effects of A77 1726 on cytokine (interleukin (IL)-8, IL-10, IL-11 secretion and tumor necrosis factor-alpha soluble receptor I (sTNFRI)) shedding in human RA fibroblast-like synoviocytes (FLS). At 100 microM, we observed an increase in IL-10 secretion, a decrease in IL-11 release and no effect on sTNFRI shedding and IL-8 secretion in IL-1beta-stimulated human RA FLS. Furthermore, at this dose, our results also confirmed that A77 1726 decreased IL-6 and prostaglandin E2 (PGE2) synthesis while it increased IL-1 receptor antagonist secretion (IL-1Ra). The mitogen-activated protein kinases (MAPKs) represent an attractive target for RA because they can regulate cytokine expression. At 100 microM, the effect of A77 1726 on IL-10 and IL-11 secretion seemed to be associated with the status of p38 MAPK activation. Our results confirmed the immunoregulatory action of leflunomide in the cytokine network involved in RA pathogenesis. It could shift the balance from cytokine mediated inflammation to cytokine directed inhibition of the inflammatory process.