Daphne from the Thymelaeaceae family, featuring a 5/7/6-tricyclic ring system and usually with an orthoester group. So far, a total of 135 diterpenoids has been isolated from the species of the genus Daphne, which could be further classified into three main types according to the substitution pattern of ring A and oxygen-containing functions at ring B. A variety of studies have demonstrated that these compounds exert a wide range of bioactivities both in vitro and in vivo including anticancer, anti-inflammatory, anti-HIV, antifertility, neurotrophic, and cholesterol-lowering effects, which is reviewed herein. Meanwhile, the fascinating structure–activity relationship is also concluded in this review in the hope of providing an easy access to available information for the synthesis and optimization of efficient drugs.There are abundant natural diterpenoids in the plants of the genus  Daphne Linn., with its ca. 95 species, is the most diverse genus in the Thymelaeaceae family [Daphne have been applied for a long history in traditional treatments for aches, rheumatism, inflammation, and abortion in Asia, Africa, and Europe [Daphne mezereum L. and other Daphne species in 1970. Subsequently, an increasing number of diterpenoids have been discovered from Daphne species.The genus e family . Some ofd Europe . Yet, noDaphne, and the genus Daphne itself also acts as an important role in the discovery of phytochemical and bioactive properties of diterpenoids. The archetype of one class, daphnetoxin, was first isolated from D. mezereum, which was named after the genus Daphne. Then, daphnetoxin and its analogues have been collectively known as the daphnetoxin class. Similarly, genkwanine A from D. genkwa is the archetypical diterpenoid of genkwanines.The diterpenoids are believed to be representative components of the genus Daphne genus also contribute to the pharmacological study of diterpenoids and have been demonstrated to possess a variety of important biological activities including anticancer, anti-inflammatory, anti-HIV, antifertility, neurotrophic, and cholesterol-lowering effects [The diterpenoids from the  effects , and somDaphne. The occurrence and distribution of these diterpenoids are also discussed thoroughly, including the source species of every diterpenoid listed in a chronological order of discovery and the parts of plants which they were isolated from. Besides, detailed information on every class of diterpenoids is provided in this review. When the adequate information is given, the structure–activity relationship (SAR) is discussed.The current review provides a comprehensive coverage of all natural diterpenoids in the genus Daphne in total. Most of the natural diterpenoids occurring in the genus Daphne belong to the daphnane type featuring a 5/7/6-tricyclic ring system with polyhydroxyl groups at C-3, C-4, C-5, C-9, C-13, C-14, or C-20 and an orthoester function located at ring C, which could be further categorized into four major classes:1–83), sharing the characteristic of an epoxy ring at C-6 and C-7;6-epoxy daphnane diterpenoids (84–95), possessing a saturated ring A and a 6,7-dihydroxyl group in the ring B;genkwanines (96–97), which could be viewed as 5-deoxy-6,7-double bond daphnetoxin derivatives;resiniferonoids (98–107), featuring a saturated ring A and a macrocyclic bridge connecting C-1 in the ring A and the end of the aliphatic orthoester group  and daphnetoxins (55–83) according to the possession of an oxygen group at C-12 in the ring C [For the 6-epoxy daphnane diterpenoids, they could be further subdivided into 12-hydroxydaphnetoxins (xins 55–8 accordin108–123). As for compounds 108–112, the skeleton resembles the genkwanine one but possesses a 3-ketone group, a C-12 oxygenated substituents, and an additional 1,2-double bond, which makes it not accord with the definition of genkwanine. What attracts more attention is the unique 4,7 or 4,6-ether structure of neogenkwanine A-I and daphneodorin H (113–123), which is believed to never be found in any other daphnane diterpenoid [Nonetheless, it is worth noticing that the existing classification system could not apply to some daphnane diterpenoids (124–133) in Daphne species, instead of a caged 9,13,14-orthoester, distinguish themselves with a cyclopropane ring D, which is regarded to be closely related to the daphnane one [134 and 135) were also reported to be isolated from Daphne genkwa, featuring a characteristic 5/11/3-membered ring system.Besides daphnane diterpenoids, tigliane diterpenoids  and daphnetoxins (55–83), based on the oxygenated substituent at C-12 in the ring C.The diterpenoids from this class share the common features of a 6α,7α-epoxy and 4β,5β-dihydroxy in the seven-membered ring B. The 6-epoxy daphnane diterpenoids could be further divided into two classes, 12-hydroxydaphnetoxins , 3-deoxy derivative (54), and the ones with a 5,20-acetonide (27 and 28). The archetypal compound of 12-hydoxydaphnanetoxins is 12-hydroxydaphnetoxin (39), which was first found as a degradation product in Lasiosiphon Burchellii Meisn. [Compared to daphnetoxins, 12-hydroxydaphnetoxins have an additional oxygen group at C-12. There are more 12-hydroxydaphnanetoxins than daphnetoxins existing in the species from the ives 43–4, 3-deoxyDaphne. Compounds of this class were mainly isolated from D. genkwa, D. tangutica, and D. acutiloba and these diterpenoids were abundant in the flower buds of D. genkwa, stems, roots, and especially bark of the plants . The archetype of this class is daphnetoxin (14), which was separated from the bark of D. mezereum for the first time [D. papyracea [D. giraldii [D. tangutica [D. acutiloba [There are less compounds from the genus rst time  and lateapyracea , D. giragiraldii , D. tangangutica , and D. cutiloba  in chron84). Among these genkwanines, only genkwanine L (95) possesses a ketone function at C-3 and an oxygen-containing function at C-12  of D. genkwa [Up to now, all compounds of this class were reported to occur in . genkwa ,57,66,73E. resinifera, showing significant transient receptor potential vanilloid 1 (TRVP1) activating activity and strong irritant effect [The resiniferonoids are a group of 5-deoxy-6,7-double bond daphnetoxin derivatives, in which the A/B ring system possesses the pattern of phorbol. One of the most representative resiniferonoids, resiniferatoxin (RTX), was isolated from the dried latex of t effect ,5, and cEuphorbia genus in the Euphorbiaceae [96 and 97) obtained from the Daphne species [Resiniferonoids have a quite narrow distribution, mostly limited to several rbiaceae ,5. Two n species  are prac98–107) have an obvious feature of bridging between the C-1′ at the end of 9,13,14-orthoester group and C-1 in the ring A with a C-C bond. Meanwhile, the ring A of 1-alkyldaphnanes is usually saturated. The 1-alkyldaphnanes also has two subdivisions, 3-hydroxy and 3-acyloxy classes, depending on the oxidation degree of C-3 , which was obtained from Gnidia subcordata originally [Daphne plants. The 1-alkyldaphnanes (98–107) tend to exist mainly in the buds of D. genkwa [D. odora [Daphne genera, there appeared to be more 1-alkyldaphnane diterpenoids in the leaves and roots of the plants from the Stellera [Wikstroemia genera [The archetype of this category is gnidimacrin  remain uncategorized into four classes mentioned above, which could be divided into two major types. The skeleton of daphgenkin A–C (109–111) and yuanhuaoate B (112) resembles the one of genkwanines  feature a 4,7-epoxy-bridged structure  in ring B, which is very rare in daphnane diterpenoids, yet there is no appropriate category for these diterpenoids. These diterpenoids (108–123) were obtained from the buds of D. genkwa with only the exception of daphneodorin H (113) isolated from the leaves and branches of D. odora , there are structural variations of a 7-ketone function and a 5,6-double bond in the ring B in the others  was isolated from commercial “mezeron” bark  in 1970 and identified as a major toxic component possessing both a similar skeleton and similar sites of functions as phorbol, which was a known toxic principle in Croton species [24) with the same skeleton as daphnetoxin (56) was identified as another toxic principle of D. mezereum [However, the poisonous principles of any  species ,96 at thmezereum .Daphne genus possess a wide range of pharmacological activities including anticancer, anti-inflammatory, anti-HIV, cholesterol-lowering, neurotrophic, antifertility, skin irritant, nematicidal, and pesticidal activities. Among these biological activities, the anticancer ones have received the most attention since there remains a need for both efficient and safe anticancer drugs with novel structures.With a considerable number of modern pharmacological and chemical studies, it was demonstrated that diterpenoids from the Daphne species exhibit potent anticancer activities against various types of cancers both in vitro and in vivo [50 values ranging from 10−6–98.46 μM , yuanhuatine (47), and yuanhuaoate B (112) share the similar structure with the only exception of an unsaturated bond in the ring A, and yuanhuatine (47) among them is the least active anticancer compound [111) and yuanhuaoate B (112), it appears that the change from the 1,10- to 1,2-double bond enhances the anticancer activity [It is generally accepted that the 1,2-double bond could be a possible alkylating functionality in a diterpenoid and thus the reduction of it might make it a less active compound . Genkwadcompound ,14. The O-benzoyl-3,5-hydroxy-6,7-epoxy-resiniferonol-9,13,14-orthobenzoate, which is a 3-hydroxyl derivative of genkwadaphnin (15), showed a promising cytotoxicity against seven carcinoma cell lines, much better than genkwadaphnin (15) itself, indicating the possibility that the hydronation of a 3-ketone favors a bioactivity [84) with a 3-hydroxyl was basically inactive against A549 cells, while its 3-acylated analogues (85–87) all possessed certain cytotoxicity with the IC50 values in the range of 0.79–8.70 μM [87) was the most potent one with a 3-benzoate group at the IC50 values of 0.79–8.00 μM [In the ring A, the variation of the oxygen-containing function at C-3 influences the anticancer activity. 12-activity . However–8.70 μM , indicat–8.00 μM  suggesti30), yuanhuadine (31), yuanhuafine (32), and yuanhuapine (46) generally showed stronger cytotoxicity against A549 cell line than genkwanine A-L (84–95) possessing a 6α,7α-dihydroxyl in the same assay [100) possessed a stronger activity in seven cell lines than genkwadane C (107) [The 6α,7α-epoxy is a characteristic structure in 6-epoxy and 1-alkyldaphnane diterpenoids, and it was reported to help enhance the anticancer activity. Yuanhuacine (me assay . Additio C (107) , suggest30) showed no inhibitory activity against P-388 in vivo, while its 20-palmitate derivative gnidilatidin 20-palmitate (19) exhibited a strong inhibitory activity at dosages of 0.5–2.0 mg/kg/d, and the 20-plamitate derivative of gnidilatin was also observed to be more active than gnidilatin itself against P-388 [The 20-acyloxy group in the ring B might have an important role in affecting the anticancer activity in diterpenoids. One type of 20-acylocy groups that could favor the bioactivity is a 20-palmitate ester. Yuanhuacine (st P-388 . This fa68) with a 20-benzoate was demonstrated to inhibit various cell lines more significantly, especially HL-60 leukemia cells, A375-S2 melanoma cells, and HT-1080 fibrosarcoma cells, than orthobenzoate 2 (71) [91) possessing a 20-benzoate exhibit much stronger cytotoxicity against P388 and A549 (IC50: 13.0 and 1.60 μM) than its analogues [Genkwanine M (e 2 (71) . Similarnalogues , indicat93–94) with a 9,13,14-orthoester were more cytotoxic (IC50: 4.20–42.0 μM) than genkwanine F–G  in P-388 and A549 cells [The caged 9,13,14-orthoester is another characteristic structure in the daphnane-type diterpenoids in general. Genkwanines J–K (49 cells .92) showed potent inhibition against P388 and A549 (IC50: 4.2 and 25.0 μM), while genkwanine F (84) were moderately active with the IC50 values of 39.0–24.0 μM [92) compared to genkwanine F (84) in different cell lines [Interestingly, for a more statured skeleton, for instance, genkwanine, the caged 9,13,14-orthoester seems to reversely have a negative impact on anticancer activity. Genkwanine J (–24.0 μM . More st30) and yuanhuadine (31) showed promising inhibition against HepG2 cell line with the IC50 values in the range of 5.56 to 17.06 μM, while yuanhuafine (32) showed more limited activity at the IC50 value of 42.37 μM [Evidence showed that the presence of a long-chain fatty could enhance the inhibitory activity against carcinoma cells. Yuanhuacine (42.37 μM .31) was inactive with the introduction of 2′,3′- and 4′,5′-double bonds [33) with one more unsaturated bond in the long-chain substituent in orthoester group presents weaker bioactivity (IC50: 809.4 nM) than yuanhuadine (31) and isoyuanhuadine  in SK-BR-3 [Whether this long chain at the end of the 9,13,14 orthoester is statured or not could influence the antineoplastic activity as well. Gnidilatin was a potent antileukemia compound against P-388 in vivo; meanwhile, yuanhuacine  and yuanhuadine (31) possessing a 2′E,4′E-double bond could both inhibit SK-BR-3 proliferation more significantly (IC50: 172.6 and 61.6 nM) than their conformational isomers, respectively (IC50: 217.1 and 83.7 nM) [As for the unsaturated long-chain at C-1′, the stereochemistry matters. Yuanhuacine  and yuan24), a major toxic principle of D. mezereum, showed significant inhibitory activity in vivo against P-388 and L-1210 leukemia cell lines at dosage of 50 μg/kg in mice [16), gnididin (18), and gniditrin (20) with the closely related skeleton isolated from Gnidia lamprantha Gilg were also proved to be substantial antileukemia agents. Huratoxin (59) and simplexin (60) exhibited similar inhibitory activities against L1210 and K562 in vitro, but less active than an esterified derivative subtoxin [39) bearing a hydroxyl at C-12 in the ring C showed no antileukemia activity and esterification of it could establish the bioactivity [15), which is in essence the benzoyl derivative of 12-hydroxydaphnin (39), was found to exert both in vitro and in vivo antileukemia activity against P-388 cells [The absence of a 12-acyl group generally causes the reduction of anticancer bioactivity. Mezerein ( in mice . Gnidicisubtoxin . By compactivity . Genkwad30) showed stronger cytotoxicity against HL-60 and K562 cells (IC50: 26.81 and 16.08 μM) than yuanhuadine . Genkwadaphnin (15), with a 12-benzoate function, was also more active in antileukemia than yuanhuafine (32), as the latter showed limited inhibitory activity in HL-60 and K562 cell lines [47) was cytotoxic against human and chronic myeloid promyelocytic leukemia cell lines at the IC50 values of 17.72 and 17.54 μM [46) has not been reported to possess antileukemia property. Yuanhuacine (30) and isoyuanhuacine (21) showed moderate cytotoxicity against SK-BR-3 (IC50: 172.6 and 217.1 nM), while and yuanhuadine (31) and isoyuanhuadine (22) were more active (IC50: 61.6 and 83.7 nM) [The type of ester group at C-12 may also have an impact on the antileukemia activity. Yuanhuacine  is one of the most potent antileukemia agents both in vitro (IC50 in the range of 0.16–0.28 nM) to HL-60, K562, and CCRF-CEM cells [Daphne species [It is noteworthy that gnidimacrin  and pimelotide C (106), it seems that the one with α-CH3 (106) exhibits more significant antineoplastic bioactivities against a variety of cell lines including HeLa, MCF-7, HepG2, HCT116, A549, A375-S2, U937, HL-60, and K562, which may suggest that the stereochemistry of substituents is related to the anticancer activity as well [Interestingly, for pimelotide A ( as well .15) could oppose the protein and DNA synthesis of P-388 cells to exert both in vitro and in vivo antileukemia activities [15, IC50: 11.8 μM) and yuanhuacine  were determined to suppress Bcl-2 and Bcl-XL in a dose-dependent manner to induce apoptosis in human myelocytic HL-60 cells [It is also revealed that genkwadaphnin  showed a significant antiproliferative effect against K562 cell lines at the IC50 of 1.2 nM by activating protein kinase C (PKC) and arresting the cell cycle at G1 phase [Gnidimacrin (G1 phase .31), yuanhuadine (63), yuanhualine (64), and yuanhuagine (33) exhibited stronger antiproliferative activity against A549 cells  than the positive control ellipticine without displaying cytotoxicity against the human normal lung epithelial cell line MRC-5, with especially yuanhuacine  presenting the most potent bioactivity [30) could also be anticancer bioactive against H1993 human non-small cell lung cancer (NSCLC) cells both in vitro and in vivo by modulation of the AMPK/mTOR signaling pathway [Several 12-hydrodaphnetoxins including yuanhuadine ( pathway .64), yuanhuadine (63), and yuanhuagine (33) showed notable inhibitory effects in drug-resistant cell lines including gemcitabine-resistant A549, gefitinib- and erlotinib-resistant H292. Further research indicated that these diterpenoids were able to arrest cell cycle in the G0/G1 and G2/M phase in A549 cells by upregulating the expression of cyclin dependent kinase inhibitors P21 and P53 as well as downregulating cell-cycle regulators, for example, c-Myc and cyclin B1/cell division cycle 2 (CDC2) complex and suppress Akt/STAT/Src signaling pathway [64) was observed to have synergistic effects with certain chemotherapeutic agents  in the treatment of A549 cell line [Yuanhualine ( pathway . Additioell line .15) on hepatocellular carcinoma (HCC) cells both in vitro and in vivo with Hep3B and PLC/PRF/5 cell lines and BALB/c nude mice, respectively, the results showed that genkwadaphnin (15) suppressed growth and invasion of HCC cells both in vitro and in vivo by blocking DHCR24-mediated cholesterol biosynthesis and lipid rafts formation [30) and genkwadaphnin (15) were hepatotoxic on normal human liver cells HL-7702 in a dose- and time-dependent manner; meanwhile, the change of cell morphology and increased AST and ALT were observed as well [Wu et al. evaluated the effects of genkwadaphnin (ormation . Evidenc as well .30) was found to be an active inhibitor in both MCF-7 and MDA-MB-231 cell lines, and the preliminary mechanism of strong cytotoxicity of yuanhuacine (30) against MCF-7 was investigated further by using Western blot and flow cytometry analysis; the results suggested that yuanhuacine (30) induced apoptosis via the regulation of Bcl-2, Bax, and cleavage of PARP expression in MCF-7 cells [47) was also observed to inhibit the growth of estrogen receptor alpha (ERα)-positive cells MCF-7 (IC50: 0.62 μM) significantly compared to tamoxifen (IC50: 14.43 μM) through mitochondrial dysfunction and apoptosis in ERα-positive breast cancer cells MCF-7 caused by ERα-downregulation [Yuanhuacine , Yuanhuatine (47), and genkwanine M (68) isolated from the buds of D. genkwa displayed pronounced inhibitory bioactivity against the human melanoma cell line A375-S2 at the IC50 levels of 8.72, 9.31, and 3.62 μM, respectively [30) and yuanhuadine (31) were obtained from the dichloromethane fraction and showed potent cytotoxicity to melanoma B16 as well as A2058 cell lines, while genkwanine C (86) and genkwanine VIII (118) were selectively active in the A2058 cells [30), yuanhuadine (63), genkwadaphnin (15), genkwanine A (84), genkwanine F (89), genkwanine H (91), and daphneresiniferin A (96) and B (97) could inhibit the α-MSH-induced melanin production in B16 melanoma cells remarkably with the IC50 in the range of 0.57–9.0 μM in comparison with the positive control arbutin (IC50: 140 μM) and kojic acid (IC50: 39 μM), especially yuanhuadine  with the striking inhibitory activity [97) showed obvious cytotoxicity against B16 cells at the IC50 level of 6.6 μM [15) was also investigated to exert apoptosis-triggering effect in squamous cell carcinoma (SCC) cells in a JNK-dependent manner [Yuanhuacine  cell and cytotoxic T lymphocyte (CTL) [Besides, the antiproliferative activity of the ethyl acetate and aqueous extract from the leaves of 10 cells ; furtherte (CTL) , hopefulD. mucronata was evaluated to possess anticancer property both in vitro and in vivo and its mechanism, similar to the natural anticancer drug Taxol, was probably related to the downregulation of human tumor necrosis factor alpha receptors (TNF-αR) [54), was isolated from D. mucronata afterwards and showed a strong antiproliferation effect on WEHI-164 by mediating the progress of DNA synthesis [The alcohol–water extract of the aerial parts of (TNF-αR) ; the proynthesis .15) was revealed when it was found that it enhanced the p21 expression and simultaneously suppressed the c-Myc expression in a PRDM1-denpendent manner to arrest the cell-cycle progression in the human colon cancer SW620 cell line [The mechanism of anticancer effect of genkwadaphnin (ell line .30) and yuanhuadine (31) were revealed to exhibit more significant inhibitory effects on the proliferation of the COLO250 (IC50: 2–3 μM) than HT-29 cell line (IC50: 13–23 μM) [30) inhibited the HCT116 cell line by upregulating p21 expression and transcription via a p53 protein independent cascade [108), along with yuanhuacine (30) and yuanhuadine (31) obtained from the petroleum ether extract from D. genkwa, showed definite cytotoxic effects on both SW620 and RKO cell lines with the respective IC50 value of 3.0 and 6.5 μM; then, the results of further research revealed that daphgenkin A (108) inhibited SW620 cell proliferation by stalling the cell cycle at G0/G1 phase, causing cell death by apoptosis as well as inducing cell cycle arrest via regulating the PI3K/Akt/mTOR signaling pathway [Yuanhuacine (3–23 μM) , suggest cascade . Daphgen30), yuanhuadine (31), yuanhuatine (47), genkwanine F (89), genkwanine N (69), and wikstroemia factor M1 (72) obtained from D. genkwa were moderately cytotoxic with IC50 levels in the range of 25.61 to 27.32 μM against the human gastric carcinoma MGC-803 cell line [30, IC50: 17 μM) and yuanhuadine  were detected to oppose the proliferation of human gastric adenocarcinoma AGS cell lines, along with genkwanine VIII  [Yuanhuacine (ell line . Further: 12 μM) .30) and yuanhuajine (7) presented more obvious inhibitory activity against DNA topoisomerase I (DNA topo I) at the IC50 levels of 40.0 and 38.3 μM than a known topo I inhibitor hydroxycamptothecin , and further study with the prepared derivatives suggested that less electron-withdrawing groups at C-5, C-12, and C-20 facilitated the combination between the compounds and DNA topo I [Yuanhuacine (A topo I .30) was also reported to be cytotoxic against two bladder cancer cell lines UMUC3 (IC50: 1.89 μM) and T24T (IC50: 1.83 μM), and it functioned in T24T cells by inducing a G2/M phase arrest significantly via modulation of Sp1 protein expression [Yuanhuacine (pression .50), yuanhuacine (30), and yuanhuatine (47) with the IC50 levels of 11.62–12.35 μM [54) showed a stronger cytotoxicity (IC50: 1 μM) [The human lymphoma cell line U937 has been reported to be moderately suppressed by daphnane-type diterpene ester-7  .D. altaica was also found to significantly suppress the proliferation of esophageal squamous carcinoma Eca-109 cell line at the IC50 level of 10.6 μM and in a dose-dependent manner [D. altaica was reported to function by inducing apoptosis and cell cycle arrest in the S phase in the Eca-109 cell line via the PPARγ-mediated pathway [The chloroform extract from t manner , and the pathway . HoweverDaphne species were demonstrated to possess an anti-HIV activity even stronger than some anti-HIV agents such as 3′-azido-3′-deoxythymidine (AZT), and structure-activity relationship in anti-HIV bioactivity is quite similar to that in the anticancer one (Both daphnane- and tigliane-type diterpenoids from ncer one .11–12), presented stronger anti-HIV activity than daphneodorins F–H  without one (EC50 > 25 nM) [35) and its isomer, isoyuanhuadine (22), showed promising anti-HIV activity (EC50 < 0.9 nM), while yuanhuaoate C (53) without a 9,13,14-orthoester merely exhibited moderated activity [The presence of a 9,13,14-orthoester in the ring C is also favorable for anti-HIV activity as diterpenoids with an orthoester, for instance, daphneodorins D–E  from D. aurantiaca showed definite anti-HIV-1 activity against C8166 cell line with EC50 value of 0.282 nM and SI value of 65177.305, while phorbol 13-monoacetate (130) possessing a 12-hydroxyl showed limited activity, which suggests an acyl group at C-12 might favor anti-HIV-1 bioactivity [The substituent at C-12 in the ring C acts as an important role in anti-HIV activity as well. A tigliane diterpenoid 12-activity .102–103) presented more potent activity in inhibiting HIV-1 replication in MT4 cell line  than daphneodorin C (EC50: 2.9 nM) suggesting that a 20-benzoyloxy reduces the anti-HIV activity [Daphneodorins A–B (activity .129) is known as a potent anti-HIV agent [98), a HIV-latency-reversing compound that is strikingly 2500-fold more potent that prostratin (129), functioned by regulating various signaling pathways including the MAPK, PI3K-Akt, JAK-Stat, TNF, and NF-κB ones [Prostratin , genkwanine N , genkwadaphnin , kirkinine , excoecariatoxin , and 14′-ethyltetrahydrohuratoxin  were obtained from D. acutiloba, all of which exhibited strong anti-HIV-1 bioactivity, especially genkwanine N  [Acutilobins A–G , gnidicin (16), gniditrin (20), and excoecariatoxin (58) were determined to be the principles of anti-HIV bioactivity according to the HPLC-based profiling; meanwhile, a more detailed study showed that daphnetoxin (56) selectively interfered with the expression of two key cell-surface factors CXCR4 and CCR5 for HIV-1 entry [Based on the discovery of the antiretroviral activity of the dichloromethane extract of -1 entry .77) inhibited not only HIV-1 but HIV-2 strains in a concentration-dependent manner with high SI values and low cytotoxicity, and its mechanism might be related to the inhibition of HIV-1 reverse transcription and integrase nuclear translocation [It is worth noting that wikstroelide M (location .15), gniditrin (20), and yuanhuatine (47) showed significant inhibitory effect against LSP-induced nitric oxide (NO) production in RAW 264.7 macrophages with the IC50 values of 0.03–0.07 μM, especially 12-O-benzoylphorbol-13-nonanoate (124) and 12-O-benzoylphorbol-13-octanoate (85) with the IC50 values of 0.01 μM could be potential therapeutic agents for inflammation [132, IC50: 7.2 μM), a tigliane-type diterpenoid derived from D. genkwa, and orthobenzoate 2  showed stronger inhibitory activity than aminoguanidine (IC50: 17.4 μM) on NO production in RAW 264.7 cells [Genkwadaphnin , the mechanism includes the activation of PKD1/NF-κB signaling to induce CD44 expression in a time- and concentration-dependent manner and thus promoting the migration of K562 cells, resulting in an innate immune response [15) was also observed to restore exhausted LCMV-specific CD4+ and CD8+ T cells by downregulating negative regulatory molecule Tim-3 [As for another inflammatory inhibitor genkwadaphnin (response . Genkwadle Tim-3 .128), genkwadaphnin (15), isoyuanhuacine (21), yuanhuacine (30), yuanhuaoate C (53), yuanhuagine (33), isoyuanhuadine (22), and yuanhuadine (31) in LPS-induced RAW264.7 cells by downregulating the overexpression of IL-6, IL-1β, and TNF-α as well as decreasing NO production as the results of principal component analysis (PCA) and hierarchical cluster analysis (HCA) indicated, among these, prostratin Q (128) also showed bioactivity on VEGF, MMP-3, and ICAM, which has the potential to be developed as a novel drug for rheumatoid arthritis treatment [It is reported that diterpenes showed anti-inflammatory efficacy sorted from highest to lowest as follows: prostratin Q (nhuadine  in LPS-i20) and daphnetoxin (56) extracted from D. giraldii were found to present potent cholesterol-lowering activity in vitro at the EC50 levels of 0.59 and 4.3 μM, respectively, by up-regulating the low-density lipoprotein receptor (LDLR) level and consequently promoting LDLR expression [20) had a lower EC50 for activating LDLR-promoter than that of daphnetoxin (56), which might indicate that the acyl group at C-12 might improve the cholesterol-lowering bioactivity as well.Gniditrin (pression . Gniditr30) and genkwanine N (69) significantly enhanced the function of the orphan nuclear receptor Nurr1 at a concentration of 0.3 μM and inhibited LPS-induced neuroinflammation in vitro as well as improved behavioral deficits in a hydroxydopamine (6-OHDA) -induced rat model of Parkinson’s disease [Yuanhuacine ( disease .133), a tigliane diterpene from the EtOAc extract of D. holosericea, was discovered to be a moderate acetylcholinesterase (AChE) inhibitor at a concentration of 100 μM, which suggested its potential usage in the mediation of Alzheimer’s disease [Dapholosericin A ( disease .134) and B (135), two lathyrane-type diterpenes isolated from the chloroform extract from D. genkwa, were demonstrated to inhibit LPS-induced NO production in microglial BV-2 cells moderately (IC50: 43.08–46.77 μM) with nearly non-cytotoxicity, while yuanhuapine (46), yuanhuatine (47), genkwanine M (68), genkwanine N (69), yuanhuafine (32), and genkwadaphnin (15) showed much stronger anti-neuroinflammatory effect with the IC50 value below 0.44 μM, affecting cell viability slightly (17.5%–32.5% cell death at 10 μM) [Genkwalathins A (t 10 μM) .D. genkwa are also used as a traditional Chinese herbal remedy for abortion. Both yuanhuacine (30) and yuanhuadine (31) have already been used clinically as labor-induced drugs at the per capita dose of 70–80 and 60 μg, respectively [47) [26) [The flowers of ectively . Yuanhuaely [47)  and tang47) [26)  also sho47) [26) , and the47) [26) .59), mezerein (24), simplexin (60), and pimelea factor P2 (99), are highly skin irritant and toxic, thus considered as toxic principles in Daphne species [Many naturally occurring daphnane and tigliane diterpenoids, including huratoxin  was isolated from the seeds of D. mezereum and verified to be skin irritant just as daphnetoxin (56) [55), daphnetoxin (56), and 12-hydroxydaphnetoxin (39) obtained from D. giraldii were discovered to exert skin irritant activity [59), genkwadaphnin (15), prohuratoxin (77), and 1,2α-dihydrodaphnetoxin (73) were isolated from D. feddei, all of which have been determined to be skin irritant, and Huratoxin (59) exhibited the highest irritant activity among these four compounds [Mezerein (xin (56) . Daphnegactivity . Huratoxompounds .D. odora was nematicidal to Aphelenchoides besseyi; then, further study showed that odoracin (25) had nematicidal activity towards A. besseyi was 100% (5 ppm) after 5 days [56) was reported to be a major poisonous component in D. mezereum [25), along with odoratrin (45), gniditrin (20), 12-O-(E)-cinnamoyl-9,13,14-ortho--decadienylidyne-5β,12β-dihydroxyresiniferonol-6α,7α-oxide (41) and 12-O-(E)-cinnamoyl-9,13,14-ortho--decatrienylidyne-5β,12β-dihydroxyresiniferonol-6α,7α-oxide (40), was found to have ornithine decarboxylase (ODC)-inducing activity in mouse skin, which correlated with a tumor-promoting effect; these compounds were simultaneously determined to be piscicidal [It has been discovered that the benzene extract from the roots of r 5 days . Daphnetmezereum . Odoraciscicidal . Daphne species, daphnetoxin (56), was isolated as a toxic principle [Daphne after decades. These diterpenoids could be classified as daphnane-, tigliane-, and lathyrane-types according to the oxygen-containing functions and substitution pattern, and daphnane-type could be subdivided into 6-epoxy, genkwanine, 1-alkyldaphnane, and resiniferonoid. Within more and more novel diterpenoids isolated from Daphne genera, some of them could not fall into any classification. For example, genkwanine L (95) with a 3-ketone and 12-acetyl group does not completely satisfy the definition of genkwanines. As for compound 108–123, one or more suitable classifications have not been established yet.Back to 1970, the very first diterpenoid from rinciple . A totalDaphne embraces more than ninety species distributed in Asia, Africa, and Europe [Daphne species and diterpenoids have been reported to occur mainly in D. genkwa. For D. mucronata, only two isolated diterpenoids but one of those, gnidilatimonoein (54), showed potent anticancer activity [Daphne. The distributions of every diterpenoid in different parts of plants are available in the hope that it would be a guide for isolation and identification of diterpenoids.Although the genus of d Europe , the oriactivity ,122,123,Daphne species have been assayed and found to possess pharmacological activities which might be closely related to diterpenoids. The methanol extract of D. malyana showed significant antimicrobial potential [D. linearifolia, of which the stem bark was used to treat inflammation and rheumatism such as some Daphne species mentioned above, showed affinity towards a promising anticancer target Hsp90 [D. cneorum extract exhibits potent antimicrobial and antioxidant activities, which makes it a possible source of new agents [D. alpina, its antioxidant and antimicrobial bioactivities have been investigated [20) has been isolated from this species [Daphne species.Furthermore, some otential  and the et Hsp90 . The D. w agents . For D. stigated ,128 and  species . These sFor the structure–activity relationship (SAR) summarized in this review, despite the fact that some of the SAR remains unclear and needs to be further investigated, it showed that the pharmacological effect, to a large extent, depends on the substituents at C-3, C-12, C-20, and C-1′ and the saturation of the ring A in these molecules. The SAR study is believed to provide very useful information for the optimization and synthesis of diterpenoids and lead to the development of novel agents; however, the SAR of diterpenoids has not been completely clarified.Daphne were covered. Many of the Daphne species are bioactive and thus have been used as traditional treatments for various diseases. Both the source species and parts from which diterpenoids were isolated have been provided as detailed reference information. The natural diterpenoids from Daphne species present interesting structures with complicated stereochemistry, which are closely related to their abundant bioactivities. The biological activities and the structure–activity relationship of certain classes of diterpenoids were reviewed in the hope of providing an easier way for researchers to understand the general situation of phytochemical and pharmacological properties in diterpenoids from the genus Daphne.In this review, a total of 135 natural diterpenoids in the past decades from the plants of the genus


Platinum chemoresistance results in high-grade serous ovarian cancer (HGSOC) disease recurrence. Recent treatment advances using checkpoint inhibitor immunotherapy has not benefited platinum-resistant HGSOC. In ovarian cancer, DNA methyltransferase inhibitors (DNMTi) block methylation and allow expression of silenced genes, primarily affecting immune reactivation pathways. We aimed to determine the epigenome and transcriptome response to sequential treatment with DNMTi and carboplatin in HGSOC.In vitro studies with azacitidine or carboplatin alone and in sequential combination. Response was determined by cell growth, death and apoptosis. Genome-wide DNA methylation levels and transcript expression were compared between untreated and azacitidine and carboplatin sequential treatment.Sequential azacitidine and carboplatin significantly slowed cell growth in 50% of cell lines compared to carboplatin alone. The combination resulted in significantly higher cell death in 25% of cell lines, and significantly higher cell apoptosis in 37.5% of cell lines, than carboplatin alone. Pathway analysis of upregulated transcripts showed that the majority of changes were in immune-related pathways, including those regulating response to checkpoint inhibitors.Sequential azacitidine and carboplatin treatment slows cell growth, and demethylate and upregulate pathways involved in immune response, suggesting that this combination may be used to increase HGSOC response to immune checkpoint inhibitors in platinum-resistant patients who have exhausted all currently-approved avenues of treatment.The online version contains supplementary material available at 10.1186/s12885-022-09197-w. Standard treatment for high-grade serous ovarian cancer (HGSOC) is cytoreductive surgery followed by a combination of platinum (carboplatin or cisplatin) and taxane (paclitaxel) chemotherapies. Although initial recurrences are often platinum-sensitive, development of resistance to platinum-based chemotherapy usually occurs with subsequent recurrences .When chemoresistance occurs, the disease-free period between recurrences progressively gets shorter. The most recent definition of platinum-resistant disease is disease recurrence within 6 months of completion of first-line platinum-based chemotherapy . The aveRecent advances in the treatment of many solid tumours using checkpoint inhibitor (CPI) immunotherapy targeting the CTLA4 and PD1–1/PDL-1 axes held great promise for HGSOC, which is often characterised by increased tumour-infiltrating lymphocytes (TILs), a biomarker of response to CPIs. Unfortunately, the results of both monotherapy and combination CPI immunotherapy has not significantly increased progression-free or overall survival for HGSOC , 15. TheCurrent evidence indicates that when low-doses of DNMTi are given, methylation is blocked but the dose is not cytotoxic –19. In oTherefore, the aim of the study was to investigate if sequential DNMTi and platinum-based chemotherapy on HGSOC can increase expression of transcripts associated with immune responses, whilst occurring in parallel to decreased proliferation and/or increased cell death, therefore priming platinum-resistant HGSOC for increased response to CPI immunotherapy.BRCA status of each cell line have been described  [Eight ovarian cancer cell lines were used in this study; KURAMOCHI, OVSAHO, COV362, OVCAR4, COV318, TYKNU, OVKATE, and OAW28. These cell lines were confirmed by genomic and proteomic profiling to be representative of HGSOC . The BRCildtype) , 21.2. Azacitidine (Sigma-Aldrich) and carboplatin (Sigma-Aldrich) were resuspended at 1 mM and 27 mM (10 mg/mL) respectively. All cell lines were treated with 0.5 μM azacytidine alone, 20 μM carboplatin alone and sequential combination of 0.5 μM azacitidine and 20 μM carboplatin. For azacitidine treatment, cells were treated for 72 h with 0.5 μM azacitidine, with media and azacitidine replaced every 24 h. For carboplatin treatment, cells were treated with 20 μM carboplatin for 48 h. For sequential combination azacitidine and carboplatin treatment, cell lines were first treated with 0.5 μM azacitidine for 72 h followed by 20 μM carboplatin for 48 h.The cell lines were cultured in high glucose DMEM (10% FBS) (Gibco). All cells were incubated at 37 °C with 5% COThese doses of carboplatin (20 μM) and azacitidine (0.5 μM) used were lower than maximum plasma concentrations of each drug when used as chemotherapy agents , 23. A mIncuCyte® Cytotox Green was used to stain dead cells after treatment and apoptotic cells were stained with IncuCyte® Annexin Red reagents. The quantification of each population of cells was performed using IncuCyte® ZOOM (Essen BioScience). The eight cells lines were seeded in 96-well plates in triplicate wells at 20% confluency; 8000 cells/well for each cell line, 10,000 cells/well for TYKNU; and the doubling time for the cell lines ranged from 40 to 96 h. They were incubated for 24 h before treatment was added (dosage and timing described above) with Cytotox Green  and Annexin Red  per manufacturer’s protocol.2 ± SEM. Cell confluence was used as a surrogate marker of cell growth in the live-cell assay.The wells were imaged using IncuCyte® Zoom live imaging system at 10x objective using the green and red channels . Phase contrast and dual-colour fluorescence images  were collected for all experiments with spectral unmixing set as 8% of red removed from green) every 6 h for 7 days. The average live (cell confluence), dead (green fluorescence) and apoptotic (red fluorescence) cell count was determined from the replicated wells and data was expressed mean (green or red) confluence per mmThe normalised cell death and apoptosis was obtained by dividing the green or red fluorescence area over the phase confluence area  cells / confluence of total cells; normalised cell apoptosis = confluence of apoptotic (red) cells / confluence of total cells).The processing definitions for analysing the confluences of phase and green and red fluorescence are described in Additional File Cell pellets were collected after 5 days of no treatment (untreated) or 5 days of sequential combination treatment (72 h with azacytidine and 48 h with carboplatin) from one independent experiment.Cell pellets for DNA extraction were resuspended in 200 μL 1x phosphate buffered saline (Invitrogen). DNA was then extracted using the Zymo Quick-DNA Miniprep Kit (Zymo Research) per manufacturer’s protocol. DNA was quantified using the Qubit™ DNA Broad Range Assay Kit (Invitrogen) and purity assessed by 260/280 ratios (> 1.8) using the Epoch Microplate Spectrophotometer (BioTek). DNA integrity was analysed using the TapeStation and D1000 ScreenTape Assay. Cell pellets with a minimum of 250 ng genomic DNA were used for methylation array.Total RNA was extracted by TRIzol (Invitrogen) extraction method according to standard laboratory protocol. RNA was quantified using the Qubit™ RNA High Sensitivity Assay Kit (Invitrogen) and purity assessed by 260/280 ratios (> 1.8) as above. RNA integrity was analysed using the TapeStation and RNA ScreenTape Assay (Agilent Technologies). Cell pellets with a minimum of 1 μg total RNA and RNA Integrity Number (RIN) ≥8 were used for RNA sequencing.In total, four HGSOC cell lines, COV362, OVCAR4, TYKNU, and OAW28 fulfilled the minimum requirements for methylation array and RNA sequencing to be performed. Additionally, these 4 cell lines show the most significant changes in cell confluence and relative cell death and apoptosis compared to standard-of-care platinum treatment . Genomic DNA extracted from cell lines as above were bisulfite-treated using the EZ DNA Methylation Kit (Zymo Research). Genome-wide DNA methylation profiles were obtained using the Infinium MethylationEPIC Beadchip (Illumina), which interrogates over 850,000 methylation sites across the genome.P-value> 0.01, non-CpG probes, and SNP-related probes were removed from the analysis. Probes on the X and Y chromosomes were excluded from the analysis. Normalisation and background correction were applied to the methylation data using PBC within the ChAMP package.Data preprocessing and analyses were performed in the statistical programming environment R v3.6.2 with the package Chip Analysis Methylation Pipeline for Illumina HumanMethylation450 and EPIC (ChAMP) v3.11 . Probes P values for multiple hypothesis testing, false discovery rate (FDR) was calculated using Benjamini-Hochberg correction, and methylation sites in CpG sites/regions with FDR-adjusted P-value< 0.275 were considered statistically significant.Differentially methylated probes (DMPs) were identified using the package within ChAMP, which calculated the difference in β-value between the untreated and azacitidine and carboplatin-treated groups. The β-value is the ratio of intensities between methylated and unmethylated alleles, with β being between 0 (unmethylated) and 1 (fully methylated) . The DMPhttp://great.stanford.edu/) was used to analyse the functional significance of differentially methylated regulatory regions [The Genomic Regions Enrichment of Annotations Tool (GREAT) ( regions .Whole transcriptome sequencing (RNA-Seq) was performed by AGRF. Total RNA was extracted from cell lines and quality of the total RNA evaluated as above. Briefly, the ribosomal RNA (rRNA) was removed from the extracted total RNA by Illumina Ribo-Zero Plus rRNA Depletion Kit (Illumina), and strand-specific RNA-seq libraries were built by TruSeq Stranded Total RNA (Illumina) according to the manufacturer’s protocol. Quality control of the libraries was performed by Agilent 2100 Bioanalyzer (Agilent Technologies). RNA sequencing was performed on the Illumina NovaSeq 6000 platform (Illumina), and 100 bp paired-end reads were generated, with a sequencing depth of 30-60 M reads. This is sufficient for characterisation of gene expression levels.The RNA-Seq reads were first trimmed with Cutadapt to remove adapter sequences, primers, poly-A tails and low quality reads. The reads were aligned to a reference genome GRCh37/hg19 using the sequence alignment tool HISAT2. Counts for mapped reads for genomic features such as genes, exons, promoters, gene bodies, genomic bins, and chromosomal locations were created using featureCounts.Differential expression analysis between the untreated and treatment groups was performed using the DESeq2 package v3.12. The estimation of transcript abundances is based on normalised base counts from all samples, dividing by size factors. The biological processes altered by gene expression changes between the groups were assessed using Ingenuity Pathway Analysis (IPA). The RNA sequencing data is available for download at the GEO repository (GSE169617).Cellular confluence was measured to determine if ovarian cancer cell growth is affected by combined azacitdine and carboplatin. The sequential combination of azacitidine and carboplatin significantly slowed the growth of all cell lines Fig. . Cells tBRCA mutations  Fig. A. This wons Fig. B. In BRCnly Fig. C.Fig. 2Pp < 0.05). For KURAMOCHI, COV362, OVCAR4, and TYKNU, confluence was significantly lower when treated with combined azacitidine and low-dose carboplatin compared to a single treatment of higher dose carboplatin . For TYKNU, combination azacitidine and carboplatin showed significantly higher cell death compared to the higher dose of carboplatin (30 μM)  or higher-dose 30 μM) carboplatin only Fig. A. This wons Fig. B and C. 0 μM carbBRCA-wildtype cell lines . One cell line, OVCAR4 had significantly higher cell apoptosis after combined azacitidine and carboplatin compared to higher-dose of 30 μM carboplatin. Two cell lines, COV318 and OAW28 had significantly higher cell apoptosis when treated with combined azacitidine and carboplatin compared to treatment with low-dose carboplatin (20 μM)  Fig. A. This wnes Fig. B. In BRCtin Fig. C. All ceP > 0.01 in more than one sample and probes that are not represented by a minimum of three beads on the array.To investigate the potential for sequential azacitidine and carboplatin to induce biological pathways related to CPI sensitivity, global demethylation was assessed in untreated cells compared to cells treated with sequential azacitidine and carboplatin. The reference genome used in all analyses was GRCh37/hg19. Probe filtering resulted in 711,405 probes after removing those that failed with a detection P < 0.05 after multiple comparison correction FDR = 0.275 was considered significant. In total, there were 86,979 DMPs found to be significantly different between the untreated and sequential azacitidine and carboplatin groups  ≥ 0.1).Differential methylation was assessed in CpG sites (DMPs); p = 3.55e-37), the main mechanism of programmed cell death after treatment with carboplatin . 36 annotated genes were upregulated after combination treatment compared to the untreated group . Analysis by IPA was performed to investigate the pathways significantly upregulated by the combination azacitidine and carboplatin treatment. One of the most significantly upregulated pathways after combination treatment was the apoptosis signaling pathway (p = 1.03e-02), which is triggered to respond to carboplatin-induced DNA damage , TNFR1 signaling, (p = 2.5e-03), death receptor signaling (p = 8.72e-03)), which are involved in response to chemotherapy. Also of note was the enrichment of the antigen presentation pathway, which is essential in presenting neoantigens or foreign peptide antigens to trigger T cell immune response [Three of the most highly enriched were pathways related to apoptotic programmed cell death . The network constructed from the ten upregulated genes indicated that the majority of changes were in pathways relating to immune responses, including cytotoxic T lymphocyte-mediated apoptosis (p = 1.44e-02), antigen presentation pathway (p = 1.65e-02), CTLA4 signaling in cytotoxic T lymphocytes (p = 3.79e-02), and PD-1 and PD-L1 cancer immunotherapy (p = 4.42e-02) , which is involved in many of these immune-related pathways.An integrated methylome-transcriptome analysis was performed to examine the association between CpG methylation and gene expression in the tumour cells after combination treatment. The DMPs obtained from the TSS1500 and TSS200 regions were selected for subsequent analysis as promoters and their surrounding regions are primarily responsible for gene expression regulation. We focused on promoter CpG island-containing genes that were simultaneously hypomethylated and upregulated after combined azacitidine and carboplatin treatment, and ten genes were identified  class I after sequential azacitidine and carboplatin treatment of HGSOC cell lines.The integrated methylome-transcriptome analysis also showed hypomethylation and upregulation of β2m after combined azacitidine and carboplatin. β2m is a component of MHC class I heterodimers responsible for antigen presentation, which shuttles peptide fragments to the cell surface for recognition by CD8+ T lymphocytes , 37. In + and CD8+ PD-1-positive tumour-infiltrating T-lymphocytes in the tumour microenvironment compared to the untreated tumours, suggesting an enhanced response to PD-1 inhibition [+ and CD8+ T-cells [Recent advances in the treatment of solid tumours using CPI immunotherapy has only resulted in modest improvement to overall response rates in HGSOC. Two clinical trials of CPI immunotherapy in patients with platinum-resistant recurrent ovarian cancer showed objective response rate of only 9.6% (12/25) for anti-PD-L1 and 9% (11/97) for anti-PD-1 , 40. Prehibition . A recen T-cells , indicat T-cells . Our hypThe results from this study are consistent with our previous study in melanoma, in which the sequential combination of a DNMTi and platinum chemotherapy induced greater cell death and slowed cell proliferation . SeveralThere are limitations to our cell line model-based study, which lacks tumour heterogeneity and microenvironment. It would be beneficial to replicate this study in an in vitro 3D cultures from primary tumours, ascites, or metastases, especially from platinum-resistant patients, to model the ovarian cancer tumour microenvironment . AdditioThe results of this study indicate that sequential azacitidine and carboplatin can slow tumour cell growth, and sequential combined low doses of azacitidine and carboplatin can slightly increase HGSOC cell death compared to clinically-relevant dose of carboplatin, which is an important outcome especially in platinum-resistant disease. The combination of sequential azacitidine and carboplatin treatment demethylates and upregulates pathways that are involved in immune response and immune checkpoint blockade. Taken together, the results suggest that the addition of azacitidine to standard-of-care carboplatin can be used in increase HGSOC response to immune checkpoint inhibitors.Additional file 1: Supplementary Table 1: Suggested processing definitions using the Incucyte® Zoom.Additional file 2.Additional file 3.Additional file 4.Additional file 5.Additional file 6.


Zataria multiflora (ZMSLN) enhanced the anticancer efficacy of the essential oil against breast cancer (MDA-MB-468) and melanoma (A-375) cells.The cancer burden is rising rapidly worldwide, and it annually causes about 8.8 million deaths worldwide. Due to chemical drugs’ side effects and the emergence of resistance, the development of new green drugs has received much attention. We aimed to investigate whether solid-lipid nanoparticles containing essential oil of ZMSLN was prepared by the high-pressure homogenizer method; particle size 176 ± 8 nm, polydispersity index 0.22 ± 0.1, entrapment efficiency 67 ± 5%. The essential oil showed a dose-dependent antiproliferative effect on MDA-MB-468 and A-375 cells at all examined concentrations . Interestingly, after treating both cells with 75 μg/mL of ZMSLN, their viabilities were reduced to under 13%.The finding showed that ZMSLN had a distinct antiproliferative efficacy; it could thus be considered a green anticancer candidate for further in vivo and in vivo studies. Cancers have many financial, emotional, and physical burdens in all societies and comprise approximately 17% of global deaths . CardiovZataria multiflora Bioss, the Lamiaceae family, is one of the most important medicinal plants in Pakistan, Afghanistan, and southern Iran; it is applied as an antiseptic and antitussive in respiratory tract disorders [Z. multiflora essential oil (ZMEO) also possesses many biological activities, including anticancer and antioxidant effects [Bone marrow depression, extreme fatigue, alopecia, loss of self-esteem or immunity, and a remarkable decline in white blood cell count are common side effects of synthetic or semisynthetic anticancer drugs , 8. Thusisorders . The Z.  effects , 12. EOs effects , 14. Sol effects , 16. We MDA-MB-468 (ATCC HTB-132 and A-375 (ATCC CRL-1619) as breast cancer and melanoma cell lines were purchased from Pasteur Institute of Iran. Stearic acid, tween 80, span 60, phosphate-buffered saline (PBS) tablets, penicillin-streptomycin, trypsin, dimethyl sulfoxide (DMSO), absolute ethanol (99.8%), and MTT -2, 5diphenyltetrazolium bromide) were bought from Merck Co. (Germany). Shellmax (China) and Gibco (USA) supplied cell culture medium (RPMI 1640) and fetal bovine serum (FBS). ZMEO was bought from Zardband Pharmaceutical Co. (Iran).ZMSLN was prepared according to our described high-pressure Homogenizer method . ZMEO . The formulation was poured into a quartz cell and situated in the hole of the device for analysis.To determine the shape of ZMSLN and free SLNs, it was first diluted two times with distilled water; one drop was then impregnated on a copper grid (200-mesh carbon-coated). Next, it was stained with phosphotungstic acid (2%) solution, dried at room temperature, and subjected to a TEM device, Philips CM30 TEM microscope (Netherlands).n = 3). ZMEO content in the supernatant was then determined by the UV-Visible method; absorbance at λ max was placed in the regression equation.ZMEO was first diluted in ethanol in a 150–350 μg/mL concentration, and absorbance was then screened (200–400 nm) to determine the maximum absorption wavelength (λ max). The standard calibration curve (with regression equation) for ZMEO was then plotted  in 96-well culture plates overnight. Wells content was then replaced with 75 μg.mL−1 RPMI complete medium (containing 10% FBS and penciling/streptomycin 1%). The cells were treated with various concentrations of ZMEO and ZMSLN , as well as free SLNs; treated plates were incubated for 24 h (37 °C and 5% of CO2 concentration). Wells’ content was then discarded and washed with PBS to the removed milky color of ZMSLN. After that, 100 μL of MTT reagent (0.5 mg/mL dissolved in RPMI) was treated for each well and incubated for 4 h at 37 °C and 5% of CO2 concentration. Finally, 100 μL/well DMSO was added to solubilization the precipitate formazan crystals to the obtained purple color. The absorbance (A) of wells was measured at 570 nm by the ELISA Plate Reader. Cell viabilities at different concentrations were calculated by eq. The in vitro cytotoxicity of ZMEO and ZMSLN on the breast cancer (MDA-MB-468) and melanoma (A-375) cancer cells was estimated by MTT assay according to our previous study with slight modification . ZMEO wa50s of the samples. One-way ANOVA  with the least significant difference test (L.S.D) at the 5% level was used to compare anticancer activities.All the tests were carried out in triplicate; results were expressed as mean ± SD. CalcuSyn software  was applied for calculating the ICDLS characterized the ZMSLN with a particle size of 176 ± 8 nm and a polydispersity index of 0.22 ± 0.1  at all concentrations. Moreover, a significant difference (P < 0.001) is observed amongst the control group and treated cells with free SLNs; viability was reduced to 79%.As shown in Fig. P < 0.001). Besides, the viability of the cell after treatment with free SLNs was significantly (P < 0.001) reduced (to ~82%) in comparison with the control group.From Fig. 50 values of ZMEO against MDA-MB-468 and A-375 cells were obtained at 380 and 59 μg.mL-1. Because even the lowest concentration of the ZMSLN reduced cell viability of both cell lines below ~ %13, IC50 of ZMSLN was thus not calculating precisely. However, it could be concluded that ZMSLN had a distinct antiproliferative effect.IC2 non-expression (triple-negative) is an invasive and poorly differentiated cell line. It is one of the commonly used cell lines in the literature [Breast and skin cancer are the two most frequent types of cancers worldwide , 20. Theterature , 24. Skiterature . Malignaterature . Amongstterature , 28.50 values were obtained as 76, 70, and 104 μg.mL−1 [50 380 μM was more potent than thymol with IC50 497 μM against human non-small cell lung carcinoma after a 24 h incubation period [Elbe et al.; thymol was more potent than carvacrol [Natural products, especially EOs, have been widely used for their therapeutic properties as chemopreventive/chemotherapeutic agents in vitro and in vivo studies –31. For  μg.mL−1 . Further μg.mL−1 . For ins μg.mL−1 –36. For  μg.mL−1 . The cytn period . RecentlCroton argyrophyllus EO with the particle size of 201.4 ± 2.3 nm and entrapment efficacy 89.63 ± 1.02% was reported; an improvement in antioxidant properties of EO in the treatment of neurodegenerative diseases was observed [Eugenia caryophyllata EO were presented a unique nanoparticulate system with a vast antimicrobial activity. The particle size was between 397 ± 10 nm and 786 ± 11, with entrapment efficacy at approximately 70% [et al. designed and prepared a SLN from ZMEO using hot homogenization and precipitation methods; the prepared SLNs had a mean size of 650 nm [et al. applied the ultrasonic Homogenizer method to produce several SLNs with different sizes [SLNs contain a hydrophobic solid matrix core with one layer of a phospholipid coating . The higobserved . In anotobserved , 44. SLNtely 70% . Anotherf 650 nm . Ehsanfant sizes .et al. prepared ZMSLN with a particle size of 255 ± 3 nm to improve and control antifungal efficiency in some fungal pathogens [Cinnamomum zeylanicum EO against Leishmania tropica and Leishmania major was increased when size was reduced to the nanoscale range, 52 ± 4 nm [50 8.94 μM) compared with free variabilin (IC50 87.74 μM) against the PC-3 cell line [Furthermore, several studies showed that formulating EO in nanocarriers leads to higher activity. For example, Nasseri athogens . In our athogens . In addi2 ± 4 nm . Also, c2 ± 4 nm . Besides−1. This is due to the maximum capacity of this nanoformulation at exanimated concentrations; with decreasing concentrations, lower efficacies become visible. In our previous report, the anticancer properties of chitosan nanoparticles containing Citrus sinensis and Citrus limon essential oils at exanimated concentrations were also not dose-dependent [−1), further research to investigate toxic effects on normal cells and other cancer cell lines in vitro and animal models are suggested.In the current study, the anticancer activity of ZMEO and ZMSLN was evaluated; results represented that ZMEO at the nanoscale size (176 nm) had a substantially more anticarcinogenic effect compared with bulk form against MDA-MB-468 and A-375 cancer cell lines. It may relate to the higher efficiency of cellular uptake and intracellular absorption of ZMSLN than the bulk form of ZMEO . Moreoveependent . HoweverZ. multiflora EO (ZMSLN) with a particle size of 176 ± 8 nm, polydispersity index 0.22 ± 0.1, and entrapment efficiency 67 ± 5% was first prepared. The comparative study showed that ZMSLN had a higher anticancer activity on tested cancer lines (MDA-MB-468 and A-375) than the EO treatment group. Furthermore, after treating both cell lines with the lowest examined concentration of ZMSLN, 75 μg/mL, their viabilities were decreased under %13. ZMSLN could thus be introduced as a green potent antiproliferative candidate. However, more studies should be evaluated to reveal the exact mechanism of anticancer activity of ZMSLN.Solid-lipid nanoparticle-containing


The cell lines used in this study  were recSNCA expression. The determination that the cells are HEK-293 rather than SH-SY5Y in origin does not impact the validity of the system or results reported in the article, including the validation of the reported assay and cell lines for identifying compounds that modulate SNCA expression. Nevertheless, readers should consider potential implications of the HEK-293 cellular context when using this system in future studies.The intention of this study was to validate cell line assays for high-throughput screening to identify compounds which lower A reviewer raised that HEK-293 cells are not expected to differentiate in response to retinoic acid as is reported in the S4 Fig legend. This figure showed that autophagy blockade increased α-synuclein levels. The authors stand by the validity of this result which has since been confirmed in other studies (e.g. [The underlying data to support all graphs reported in the article are available upon request from DRS.Daniel.Scoles@hsc.utah.edu.DPH is deceased, and so as of the time of this notice, DRS will serve as this article’s corresponding author: Department of Neurology, University of Utah, 175 North Medical Center Drive East, 5th Floor, Salt Lake City, Utah, 84132, United States of America.


However, new adjuvant techniques to induce long lasting and curative CHM1319-specific TCR-transgenic T cell-mediated anti-tumor responses are needed. In this work, we sought to identify a technique to improve the cytotoxic effect of CHM1319-specific TCR-transgenic T cell by altering the immunogenic cell surface marker expression on EwS cell lines using different cytokines. We demonstrate that TNF, IL-6, IL-1β and PGE2 cause pro-immunogenic CD83, MHC class I and II as well as ICAM-1 upregulation in EwS cell lines. This observation was associated with significantly improved recognition and killing of the tumor cells by EwS-specific CHM1319/HLA-A*02:01-restricted TCR-transgenic T cells. Conclusively, we demonstrate that the induction of an inflammatory signature renders EwS more susceptible to adoptive T cell therapy. TNF, which is upregulated during inflammatory processes, is of particular translational interest as its secretion may be induced in the patients e.g., by irradiation and hyperthermia in the clinical setting. In future clinical protocols, this finding may be important to identify appropriate conditioning regimens as well as point of time for adoptive T cell-based immunotherapy in EwS patients.Ewing’s sarcoma (EwS) is a pediatric solid tumor entity with low somatic mutational burden and a low rate of tumor-infiltrating T cells, indicating a low extent of immunogenicity. In EwS, immunogenicity may furthermore be significantly diminished by a predominantly M2 macrophage driven pro-tumorigenic tumor microenvironment. In the past, we demonstrated that CHM1 In at least one out of three patients, CHM1319-specific TCR-transgenic T cell homing to affected bone marrow could be monitored. Tumor regression was observed without causing symptoms of graft versus host disease (GvHD) [Ewing’s sarcoma (EwS) represents a highly malignant pediatric solid tumor entity. EwS carries a low somatic mutational burden and a low rate of tumor-infiltrating T cells, implying a low immunogenicity. Upregulation of the endochondral bone protein Chondromodulin-I CHM1) in EwS is driven by the fusion oncogene EWS-FLI1. In this context, CHM1 has been identified as a promising target antigen for immunotherapy in EwS patients  in EwS i,3,4. Our in vivo . In the e (GvHD) .+ T cell stimulation [+ T cells upon TCR activation which, when binding to CD83 on APC, results in increased antigen-specific CD8+ T cell numbers [CD83 is a transmembrane glycoprotein of the immunoglobulin superfamily and is predominantly expressed on several activated immune cells . Two isomulation . Further numbers .319-specific TCR-transgenic T cells in vitro.In this analysis, we hypothesized that an immunogenic signature can be induced using different cell mediators in EwS cell lines. In particular, we hypothesized an immunogenic effect of cell mediators used in the differentiation of dendritic cells out of CD14+ myeloid cells. We hypothesized that mCD83 up-regulation in EwS is feasible, causing significantly improved susceptibility of HLA-A*02:01 positive EwS cell lines when treated with CHM1+ EwS line A673 and HLA-A*02:01- EwS cell line SBSR-AKS were cultured in medium containing X-Vivo 15 and 1% human AB serum. The protocol applied to the ‘supplemented’ EwS cell lines is generally used to differentiate CD14+ monocytes into DCs [5 tumor cells was supplemented with 1000 U/mL IL-4  and 800 U/mL GM-CSF . Medium was renewed at the same concentrations on day three. On day six, in accordance to DC maturation protocols, the medium was again renewed and supplemented with IL-6 (1000 U/mL), IL-1β (10 ng/mL), TNF (10 ng/mL) and PGE2 (1 µg/mL) . Further analyses commenced at day 7 . The antibodies ICAM-1, PD-L1, PD-L2, CD83, MHC class I and II and respective isotypes were purchased from Miltenyi Biotec. Respective APC-, APC-Vio770- or PE-conjugated IgG isotypes served as staining controls. Control EwS cell lines cultivated in RPMI 1640 medium  constituted respective control groups.Surface expression of CD83 (APC-Vio770), MHC class I and II (APC), ICAM-1 (PE), PD-L1 (PE) and PD-L2 (APC-Vio770) were determined by flow cytometry analysis using MACSQuant2), respectively, or (2) three out of four DC maturation cytokines, respectively. The reason why we chose this approach was to juxtapose the possible immunogenic effect in respective contrast to the effect of the three remaining cell mediators in order to screen for the strongest impact of each single mediator. To determine the point of time of CD83 surface upregulation, we performed FACS analyses at day four, day six, and day seven of the maturation protocol  either only one of the DC maturation cytokines . For analysis of the flow cytometry data, gates were set to identify living cells as well as single cells only. Furthermore, quadrants were set according to the respective APC-, APC-Vio770- and PE isotype-labelled control samples.319/HLA-A*02:01-specific TCR transgene was introduced into T cells using the retroviral vector pMP-71 [+ T cells were activated with 3 µg/mL OKT3 according to manufacturer’s recommendation and 100 U/mL IL-2 (Novartis) was added for T cell proliferation. Two days later, spin infection with virus supernatant from the packaging cell line RD114 was performed. On the next day, T cells were re-infected with the same approach. After verifying successful transduction via FACS staining, T cell subsets were purified with anti-phycoerythrin (PE) magnetic beads (Miltenyi Biotec) coupled to CHM1319/HLA-A*02:01-specific PE labeled multimers as previously published [The CHM1r pMP-71 ,13. Puriublished . Upon ge319/HLA-A*02:01-specific recognition and killing of A673 EwS cells cultured in cytokine supplemented medium compared to the control group cultured in standard control medium, was assessed using interferon-319-specific TCR-transgenic T cells were thawed and expanded by adding 100 U/mL rhIL-2 and 2 ng/mL rhIL-15. T2 cells primed with influenza and CHM1 peptides served as negative and positive control groups, respectively. For the interferon-319/HLA-A*02:01-specific T cells and 20,000 target cells were used. For granzyme B ELISpot assays, effector to target ratios were titrated from 2.5:1 to 0.156:1. All experiments were performed in sextuplicates for interferon-CHM1® WT Expression Kit) which was fragmented and labelled following the Affymetrix® GeneChip® WT Terminal Labelling and Hybridization protocol prior to its injection onto a GeneChip® Probe Array. Alterations in gene expression were compared and analyzed by gene set enrichment analysis and visualized using iSEE (Bioconductor). Affymetrix-based RNA expression analyses were performed in order to evaluate RNA signatures induced by DC medium conditioning in A673 and SBSR-AKS EwS cell lines. Gene expression analyses were illustrated using iSEE [RNA from A673 and SBSR-AKS cells cultivated in supplemented–versus control medium was isolated using the extraction reagent TRIzol and the Zymo Research Direct-zol™ RNA MiniPrep Kit. The extracted RNA served as a template for cDNA synthesis  was used to calculate mean and standard deviation of the mean (SEM). Differences between two groups were determined using the student’s Heatmap analyses showed an increase of CD83 and ICAM-1 expression in both DC medium supplemented versus control A673 and SBSR-AKS cell lines . SBSR-AKOn day seven of cell mediator treatment , we asse2 and IL-1β treatment alone, respectively. Correspondingly, FACS analysis of A673 treated with three out of four cytokines showed a varying increase in CD83 surface expression in all groups , 0.625 (p = 0.04) and 0.156 (p = 0.03), confirming stronger anti-tumor effects when A673 was supplemented (We evaluated the effect of CD83 upregulation on the cytotoxic potential of CHM1 T cells  directedere used A. The grependent . A673 sulemented B. These lemented .According to a recent analysis performed by Gröbner et al., pediatric cancers can be genetically subdivided into two classes: entities with low mutation rates and entities with structural/copy-number variants . In the 319/HLA-A*02:01-specific T cells. Therefore, we treated EwS cell lines with different cytokines routinely used for DC generation of CD14+ monocytes. Interestingly, we observed a wide spectrum of upregulated immunogenic genes and surface markers upon treatment associated with immunogenicity towards T cells, such as CD83, ICAM-1 and MHC class I upregulation both on RNA level and protein level. Given these observations, we sought to identify the cytokine responsible for inducing such immunogenicity in EwS.In the present study, we sought to identify cytokines with the potential to render EwS more susceptible to treatment with CHM1ICAM-1, also known as CD54, is a transmembrane glycoprotein of the Ig superfamily and is most prominently known for its co-stimulatory function enhancing the interaction between DCs and T cells . ICAM-1 As indicated previously, several types of cancer downregulate their cell surface expression of MHC class I over time as an immune escape mechanism by which they dampen the immune response directed against tumor tissue . Studies+ and CD8+ T cell responses. In immature DCs, CD83 is preformed intracellularly and is rapidly expressed on cell surface upon activation and maturation [Membrane-bound CD83 is a surface protein suggested to be a key immunological player in the regulation of CD4turation ,24. Furt319/HLA-A*02:01-specific T cells in ELISpot assays.Among the implemented cytokines of the DC-supplemented EwS culture media in this study, TNF was associated with most prominent CD83 upregulation. These findings are in line with previous reports identifying TNF as an activating agent for increased CD83 expression on monocyte-derived DCs and therefore a potent driver for DC maturation ,26. More319-specific TCR-transgenic T cells.In the past, studies conducted with TNF as an anti-tumor agent have yielded heterogeneous results. As a systemic agent, simultaneous application of TNF and other anti-cancer agents resulted in an increased uptake and enhanced anti-tumor efficacy. However, TNF has also shown to induce severe toxicity at doses even lower than the predicted efficient dose ,28,29. Hh as EwS ,33. Inteh as EwS ,35. Varih as EwS ,36. ConsThe present results imply that it is possible to induce tumor immunogenicity in primarily non-immunogenic EwS via exogenous stimuli. Neo-adjuvant sensitizing of therapy-refractory EwS induced by triggering a local inflammation in the tumor area, for example by local irradiation or by delivery of inflammatory mediators, may be a further step towards the improvement of successful T cell-based immunotherapy. Rübe et al. showed that increased biologically active TNF levels can be induced in a dose-dependent manner in a majority of EwS cell lines by local irradiation in both in vitro and in xenograft mouse models . Our obs319/HLA-A*02:01-specific T cells may significantly enhance anti-EwS responses.In the present analysis, we demonstrate that EwS can be altered to become more susceptible to the effect of adoptive T cell therapy using proinflammatory cytokines conventionally used for the generation of DCs. In the future, the induction of immunogenicity prior to adoptive transfer of CHM1


Listeria effect. This manuscript evaluates the intestinal cell monolayer invasive capacity of Listeria monocytogenes strains obtained from human, salmon, cheese, and L. innocua treated with two WPP (WPP-N and WPP-C) of different polyphenol contents using Caco-2 and SW480 cells. The invasion was dependent of the cell line, being higher in the SW480 than in the Caco-2 cell line. Human and salmon L. monocytogenes strains caused cell invasion in both cell lines, while cheese and L. innocua did not cause an invasion. The phenolic contents of WPP-N are characterized by high levels of anthocyanin and stilbenes and WPP-C by a high content of phenolic acids. The inhibitory effect of the WPPs was dependent of the strain and of the degree of differentiation of the intestinal cells line. The inhibition of Listeria invasion by WPPs in the SW480 cell line, especially with WPP-C, were higher than the Caco-2 cell line inhibited mainly by WPP-N. This effect is associated with the WPPs’ ability to protect the integrity of the intestinal barrier by modification of the cell–cell junction protein expression. The gene expression of E-cadherin and occludin are involved in the L. monocytogenes invasion of both the Caco-2 and SW480 cell lines, while the gene expression of claudin is only involved in the invasion of SW480. These findings suggest that WPPs have an inhibitory L. monocytogenes invasion effect in gastrointestinal cells lines.Red wine pomace products (WPP) have antimicrobial activities against human pathogens, and it was suggested that they have a probable anti- Listeria monocytogenes is a Gram-positive pathogenic bacterium, responsible for serious systemic infections such as septicemia, gastroenteritis, and meningitis, among others, both in animal species and in humans, with a high lethality rate [L. monocytogenes is difficult due to its ability to tolerate a wide range of temperatures (−1.5–45 °C) and pH (4.3–9.1), salt concentration, and lower water activity. Furthermore, it is a facultative microorganism, resistant to alkaline media and to preservatives, such as sodium nitrate [inlA and inlB, respectively, are the main invasion factors, which bind to the receptors present on the surface of the host cell E-Cadherin and Met, allowing adherence and, therefore, the invasion of bacteria after the destabilization of the cell membrane [Microbial cross-contamination is a problem in the food industry, and listeriosis outbreaks are a focus of special interest ,2. Listeity rate ,4. Accortures −1.–45 °C an nitrate . Due to  nitrate , associamembrane ,9.To prevent microbial cross-contamination and increase foodstuff safety and shelf lives, the food industry uses antimicrobial agents—commonly, synthetic or “chemical” additives. However, in the last decade, the demand for using natural products with antimicrobial properties has increased. The activity of natural products derived from plants against microorganisms has been mainly associated with the presence of phenolic compounds with antimicrobial activities ,11. PhenL. monocytogenes has been described in previous works [Listeria activity could be attributed to the wine pomace flavonoids that interact with soluble extracellular proteins and the bacterial cell wall [L. monocytogenes intestinal invasion [Wine grapes are very rich in phenolic compounds, and most of them are not extracted during the winemaking process. Thus, the wine industry generates large amounts of grape pomace rich in phenolic compounds that have been considered as an alternative for obtaining sustainable functional ingredients, which may be used in the food industry with different purposes ,16,17, aus works . The antell wall ,20,21. Iinvasion ,23,24.Listeria monocytogenes could then be used as a functional ingredient in the food industry. Therefore, we investigated the invasion of L. monocytogenes strains treated with WPP using Caco-2 and SW480 intestinal cell lines and the expression of adheren and the tight junctions involved in the mechanism of invasion. Our hypothesis is that wine pomace products that have antimicrobial activity in vitro might also have invasion potential capacity against different L. monocytogenes strains.Wine pomace products that have antivirulence effects on different strains of v/v), iron (III)-chloride acid (FeCl3), iron (II)-sulphate (FeSO4), sodium acetate (NaC2H3O2), and sodium carbonate (Na2CO3) were purchased from Panreac Quimica S.L.U. . Malvidin-3-glucoside was obtained from Estrasynthese .Saline peptone water, Brain Heart Infusion (BHI), Tryptic Soy Broth (TSB) supplemented with Yeast, Plate Count Agar (PCA), Tryptone Sulphite Neomycin (TSN), Dextrose Sabouraud (DS), and Mannitol Egg Yolk Polymyxin (MYP) mediums were obtained from Merck KGaA, . Acetonitrile; amphotericin B solution (250 μg/mL); 2,2′-Azinobis 3-ethylbenzothiazoline-6-sulphonic acid (ABTS); Fetal Bovine Serum (FBS); glacial acetic acid; hydrochloric acid; 6-hydroxy 2,5,7,8-tetramethyl-2-carboxylic acid (Trolox); L-glutamine solution (200 mM); MEM ; methanol; penicillin ; pure phenolic compounds ; streptomycin solution ; and 2,4,6-Tris (2-pyridyl)-S-triazine (TPTZ) were purchased from Sigma-Aldrich Chemical Co. . Folin–Ciocalteu (FC) reagent, 70%  and WPP-C (red wine pomace product from the center winery), were made by applying previously described processes with minor modifications. Briefly, each collected wine pomace was dehydrated in an oven at 60 °C. Each WPP was made from seedless dried wine pomaces by milling and sieving (particle size <0.250 mm), and the microbiological stabilization was done by heat treatment (90 °C/90 min).Red wine pomaces were collected, from the same vintage, in two different wineries of two Spanish wine regions, one cited in the north and other in the center of Spain. Both wineries made red wines mainly with Clostridium spp., 1 mL was poured on PCA and TSN, respectively, and incubated at 30 °C for 72 h for TVC and 37 °C for 24 h in anaerobic conditions for Clostridium spp. One hundred microliters were streaked on DS and MYP to determine the molds and yeast and Bacillus spp., respectively, and incubated at 25 °C for 5 days for the molds and yeast and 30 °C during 24 h for Bacillus spp. Microbiological assays were carried out in triplicate.One gram of each one of the WPP (N and C) was added to 9 mL of different culture media  and incubated for 24 h at 37 °C. To determine the total viable counts (TVC) and Extractable phenolic compounds of each WPP were quantified by HPLC after extracting using acidified methanol. Rehydrated powders were extracted with methanol/HCl (97:3) at 25 °C for 24 h and continuous stirring. Then, supernatants were decanted and filtered through a VWR cellulose filter with 8–12-µm particle retention . Each supernatant was raised to a known previous volume to be analyzed. Extracts were made in triplicate. Different phenolic compounds were analyzed using analytical reverse-phase HPLC on an Agilent 1100 series HPLC system  coupled to a diode array detector. Hydroxybenzoic acids, hydroxycinnamic acids, flavan-3-ols, and stilbenes were analyzed using a spherisorb3 ODS2 reverse-phase C18 column  using the chromatographic conditions previously published . RetentiQUENCHER (Q-) methods better reproduce the real circumstances in which WPP solid products, and not their extracts, are used. Q-methods are based on the corresponding classical methods, but they were adapted to measure the TAC  of solid products. QUENCHER versions of three classical TAC assays: Folin–Ciocalteu index, ABTS , and FRAP (Ferric Reducing Antioxidant Power), were selected to evaluate the TAC of the WPP . QUENCHEL. monocytogenes strains used in this study were either previously isolated from food products in the food technology area at the University of Burgos  or from human cases  obtained from the International Life Sciences Institute (ILSI) North America Listeria monocytogenes strain collection [L. monocytogenes PCR-serogrouping was performed for the salmon-derived strain using a multiplex PCR as previously described [Listeria strain was grown on Tryptic Soy Broth supplemented with yeast extract (TSB-Y) at 37 °C overnight to obtain 9 log cfu/mL. Then, the culture was diluted to inoculate 7 log cfu/mL in BHI supplemented with 40 g/L of each WPP (WPP-N and WPP-C) and incubated for 24 h and 48 h at 37 °C. The quantity of WPP supplemented was decided considering previous satisfactory results [The llection . L. monoescribed , using Iescribed . Bacteri results . To cons2. Cell monolayers were infected with the different strains of L. monocytogenes with and without a previous treatment with WPP-N and WPP-C, and a multiplicity of infection (MOI) of 25:1 was used. After 1 h of incubation at 37 °C, the cell monolayers were washed with PBS and incubated in MEM and 10% of fetal bovine serum, containing gentamicin (100 µg/mL) for 4 h (intracellular growth). Half of the samples were lysed with 1 mL of 0.1% Triton X-100, and counting was performed by plating on TSAYE agar. The other half of the samples were tripsinized and centrifuged, and the pellets were frozen until the q-PCR analysis. All assays were independently repeated four times.For the in vitro virulence assay, human adenocarcinoma cell lines Caco-2 and SW480 from ECACC  were used, and the assays were performed as previously described . Caco-2 ® Green Supermix . All the procedures were performed according to the manufacturers’ protocols. The sequences of the primer sets (forward and reverse) were, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 5′-GCTCTCCAGAACATCATCCC-3′ and 5′-GTCCACCACTGACACGTTG-3′; claudin, 5′-GTGGAGGATTTACTCCTATGCCG-3′ and 5′-TCAAGGCACGGGTTGCTT-3′; E-cadherin, 5′-ATGCTGAGGATGATTGAGGTGGGT-3′ and 5′-CAAATGTGTTCAGCTCAGCCAGCA-3′; and occludin, 5′-TCAAACCGAATCATTATGCACCA-3′ and 5′-AGATGGCAATGCACATCACAA-3′. Amplification efficiencies were calculated for each pair of primers, and efficiency-corrected quantification was performed using 2−∆∆Ct with GAPDH as the reference gene. Relative gene expression was expressed as the folds of change compared to the control (control cells uninfected with L. monocytogenes).Caco-2 and SW480 pellets were resuspended in TRI Reagent solution . After treatment with DNase I , 1 µg of total RNA was reverse-transcribed using a First Strand cDNA Synthesis kit (Thermo Fisher Scientific), and, finally, amplified using an iQ™ SYBR® Centurion XVI, version 16.2.04 . ANOVA analysis and the Student’s t-test were used to determine the statistical significance, with a p-value of <0.05).Representative data were expressed as the mean ± standard deviation of independent experiments. Statistical analysis was performed using StatgraphicsL. innocua, showed in vitro [The antioxidant and antimicrobial properties of wine pomace products (WPP) are of interest to the food industry, and they confer the possibility of considering the role of WPP as food preservatives. In previous studies, it was observed that the WPP obtained from red wine pomace could be used as natural alternative additives due to their antioxidant activity, their ability to improve microbial stability, inhibiting spoilage bacteria growth in foods with low contents of salt , and thein vitro .L. monocytogenes treated with two types of WPP, together with their effect on intestinal E-cadherin as an InlA cell receptor and on tight junction proteins occludin and claudin for their role in the maintenance of intestinal paracellular permeability.In this study, we evaluated the possible association between the bactericide effect of WPP observed previously and their antivirulence effect in intestinal cells. Intestinal epithelial cells are the first line of defense against pathogens that can breach this barrier. The implication of cell junction proteins (adherens and tight junctions) is that they are critical for the maintenance of the intestinal barrier. Therefore, this study evaluated the intestinal invasion of L. monocytogenes virulence, and to avoid contamination from possible microorganisms contained in WPP, the microbiological quality of each WPP was evaluated. The results revealed that both WPP (N and C) did not show any microbial load.Before studying the WPP effect in Furthermore, extractable phenols were analyzed as the principal antimicrobial agents of WPP, and the antioxidant capacities were considered as an index of additional interesting properties of the WPP also associated to the phenol content.The levels of the different phenolic compounds analyzed  showed sThe largest differences were detected between the anthocyanin levels, which were significantly higher in WPP-N than in WPP-C . On the contrary, the levels of the phenolic acids (hydroxybenzoic and hydroxycinnamic acids) were statistically higher in WPP-C than in WPP-N. The total content of the flavan-3-ols was similar in both WPP, but stilbenes, like anthocyanins, were statistically higher in WPP-N than in WPP-C. These results evidenced the possible large variability that can occur between the compositions of the wine pomaces collected in different regions and wineries, which derived from pedologic-climatic conditions, cultural vine practices, and oenological processes, among other factors.The results of the total antioxidant capacity (TAC) of both WPP also highlighted the differences between the products . The Q-FL. monocytogenes can cause two forms of listeriosis: noninvasive gastrointestinal listeriosis and invasive listeriosis [L. monocytogenes strains isolated from humans ; salmon (S11); and cheese (E10.652), together with one L. innocua strain, was evaluated after their serotyping.teriosis ,2. The iThe serotyping results showed that the salmon strain belonged to the serogroup 1/2a, 3a, as well as the cheese strain and ILSI18. ILSI9 belonged to the serogroup 1/2b, 3b, 7 and ILSI17 to the 1/2c, 3c serogroup. It is well-known that serogroups 1/2a, 3a and 1/2c, 3c are related with the food environment, and 1/2b, 3b, 7 and 4b, 4d, 4e are related with human cases and outbreaks. Besides, the 1/2a, 1/2b, and 4b serotypes are responsible for 98% of listeriosis cases .Listeria strains was evaluated using two cell lines: Caco-2 and SW480, with different cell structures and growth rates. The Caco-2 cell line is a model of differentiated cells that retain their morphology and most of the enterocytes function, resembling the intestinal epithelium [The invasion capacity of the ithelium . The SW4ithelium . Cell adithelium . ChangesL. monocytogenes is due to the capacity to adhere, invade, and multiply within cells, adhesion to the intestinal epithelium being the first step in the pathogenesis that precedes the invasion, and consequently, the inhibition of adhesion can prevent colonization and can limit opportunities for systemic infection [Listeria strains’ invasive potential must also be considered [It is known that the virulence of nfection . Therefonsidered .Listeria strains was evaluated at two times of bacterial growth (24 and 48 h). Monolayer cultures of the Caco-2 and SW480 cells were inoculated with each Listeria strain cultured for 24 and 48 h. The results of the invasion produced by the different strains were expressed in cfu/mL after the lysis of the host cells. The obtained results showed the invasive capacity of ILSI9, ILSI7, ILSI18, and S11 at 24 h and 48 h in both cell lines, while E10.652 and L. innocua did not invade in either cell line  and was higher in the SW480 than in the Caco-2 cell line  among the invasion capacity of each Listeria strain, which were more numerous in the case of the more differentiated cell model, the Caco-2 cell line. ILSI17 was the most invasive Listeria strain in the two cell lines.In general, the invasion of ell line . The datL. monocytogenes invasive capacity observed between both intestinal lines could be a consequence of the different characteristics of each type of intestinal cell line. The Caco-2 cell line exhibits characteristics of enterocytes with a polarized epithelium with a homogenous distribution of microvilli. However, SW480 cells correspond to nondifferentiated cells, and their surface protein expression differs from the Caco-2 cells with irregular microvilli and with high losses of the tight junction, indicative of highly disorganized epithelia [Listeria Adhesion Proteins (LAP) binding Hsp60 activates NF-κB signaling in Caco-2 cells, facilitating the myosin light-chain kinase (MLCK)-mediated opening of the epithelial barrier via the cellular redistribution of the tight junction proteins (claudin-1 and occludin) and adheren junction protein (E-cadherin) [Listeria surface [L. monocytogenes into the intestinal barrier [L. monocytogenes incubation using L. monocytogenes strains.The difference in the pithelia . Furtheradherin) . E-cadhe surface , and thi barrier . TherefoListeria strains (E10.652 and L. innocua) did not induce any changes in the expression of TJ proteins (claudin and occludin) or of the adheren (E-cadherin) compared with the noninfected cells (control) . This fact can explain the incapacity of these strains to invade the cells (both Caco-2 and SW480) (The noninvasive d SW480) . Howeverp < 0.05)  A. Beside < 0.05) . This fa < 0.05) . The mosell line ,48, the Listeria invasive strains after their incubation with WPP-N and WPP-C. (L. innocua and E10.652 were excluded from the study due to their lack of invasion capacity). The cell invasion capacity of each WPP-treated Listeria strain was expressed as the percentage of invasion with respect to the control group (cell lines inoculated with Listeria strains grown in the absence of WPP). L. innocua, a nonpathogenic Listeria spp. found in similar environments to L. monocytogenes [Phenolic compounds are efficient bacteriostatic agents, and different studies have considered their uses as anti-invasion agents of bacteria . Hence, ytogenes , was incListeria strains . Quantitatively, the values were higher, ranging between 3.7 and 1.8 times, in the case of the gene expressions of occludin  and ranged between 2.9 and two times, respectively , keeping the cell permeability and reducing the bacteria capacity to infect the cells. It is also remarkable the inhibitory effect of the incubation with WPP-C, raising the 100% of invasion inhibition of the Caco-2 cells for the strains ILSI18 and S11 (p < 0.05), the results were also notable since this strain corresponds to the serogroup 1/2b, one of the most related to human cases.With respect to the Caco-2 cell line, both WPP, in general, induced a high inhibition of the invasion capacity A,E, alth ILSI17;  showed toccludin C. In theectively D. No sigectively B. The la and S11 E. The in and S11 G,H. Leve and S11 F. FurtheListeria strains incubated with both WPP induced high invasiveness inhibition of the four strains ; then, some levels of gene expression were higher than those of their control (L. monocytogenes strains were treated with WPP-N respect the corresponding control. These results reflect the involvement of this tight junction protein and the protection of WPP against L. monocytogenes invasion of SW480 cells. In this case (SW480 cell line) results showed a significant effect of WPP incubation on the levels of claudin. Gene expression levels of this protein were higher than the control , noting a higher level of protection, mainly in the case of ILSI17 and ILSI18 incubated with WPP-C (L. monocytogenes invasion involved their interactions on the expressions of both TJ and AJ proteins.With respect to SW480 cell line, the invasion with  strains A,E, alth control B,C,F–H. th WPP-C B,F. AfteL. monocytogenes studied strains. This fact could be due to some of the components of WPP, especially some phenolic compounds. Some previous papers reported the anti-listeria activity of various natural products, among them flavonoids [L. monocytogenes invasion. Some flavonoids as chalcone inhibited SrtA activity [L. monocytogenes [Listeria strains.Altogether, the results revealed a significant effect of WPP on the virulence of four avonoids ,50 that activity . Then, aactivity , and othytogenes . Both flL. monocytogenes, probably responding to the degree of differentiation of each cell line and the level of expression and maturation of their cell-cell junctions. In this aspect, the TJ protein occludin is involved in the L. monocytogenes invasion of both Caco-2 and SW480 cell lines while claudin is only involved in the invasion of SW480. By the other hand, E-cadherin is also implicated in this invasion, mainly in the cases of the most invasive Listeria strains and more clearly in the case of Caco-2 cell line.The data suggest that there are different mechanisms involved in the invasion of Caco-2 and SW480 cells by L. monocytogenes strains studied. The capacity of WPP to reduce or inhibit the invasion of the Listeria strains is associated with their ability to modify the gene expression of adherens and tight junction proteins, which play a notable role in maintaining the integrity of the intestinal cell membrane. Therefore, the results of this study suggest an inhibition of intestinal cell invasion by L. monocytogenes treated with a wine pomace product.Wine pomace products, with rich contents of the bioactive compounds’ polyphenols, are effective natural products against the invasion of the four invasive