N-alkylisatins, a class of potent microtubule destabilizers, are a promising alternative to traditionally used antimitotics with previous demonstrated efficacy against solid tumours in vivo and ability to overcome P-glycoprotein (P-gp) mediated drug resistance in lymphoma and sarcoma cell lines in vitro. In this study, three di-brominated N-alkylisatins were assessed for their ability to retain potency in vincristine (VCR) and 2-methoxyestradiol (2ME2) resistant ALL cell lines. For the first time, in vitro neurotoxicity was also investigated in order to establish their suitability as candidate drugs for future use in ALL treatment.Drug resistance and chemotherapy-induced peripheral neuropathy continue to be significant problems in the successful treatment of acute lymphoblastic leukemia (ALL). 5,7-Dibromo-N-alkylisatins to overcome chemoresistance. Interaction of N-alkylisatins with tubulin at the the colchicine-binding site was studied by competitive assay using the fluorescent colchicine analogue MTC. Human neuroblastoma SH-SY5Y cells differentiated into a morphological and functional dopaminergic-like neurotransmitter phenotype were used for neurotoxicity and neurofunctional assays. Two-way ANOVA followed by a Tukey’s post hoc test or a two-tailed paired t test was used to determine statistical significance.Vincristine resistant (CEM-VCR R) and 2-methoxyestradiol resistant (CEM/2ME2-28.8R) ALL cell lines were used to investigate the ability of N-alkylisatins displayed bioequivalent dose-dependent cytotoxicity to that of the parental control cell line. The N-alkylisatins proved to be significantly less neurotoxic towards differentiated SH-SY5Y cells than VCR and vinblastine, evidenced by increased neurite length and number of neurite branch points. Neuronal cells treated with 5,7-dibromo-N-(p-hydroxymethylbenzyl)isatin showed significantly higher voltage-gated sodium channel function than those treated with Vinca alkaloids, strongly supportive of continued action potential firing.CEM-VCR R and CEM/2ME2-28.8R cells displayed resistance indices of > 100 to VCR and 2-ME2, respectively. CEM-VCR R cells additionally displayed a multi-drug resistant phenotype with significant cross resistance to vinblastine, 2ME2, colchicine and paclitaxel consistent with P-gp overexpression. Despite differences in resistance mechanisms observed between the two cell lines, the N-alkylisatins are able to retain cytotoxicity towards ALL cell lines with functionally distinct drug resistance mechanisms and show potential for reduced neurotoxicity. As such they pose as promising candidates for future implementation into anticancer regimes for ALL. Further in vivo studies are therefore warranted.The  Acute lymphoblastic leukemia (ALL) is a cancer of the blood and bone marrow. It is the most common childhood cancer, and its incidence is expected to increase worldwide , 2. OverVinca alkaloid vincristine (VCR) is a standard component in every combination chemotherapy regimen used to treat ALL , where I is the macroscopic current, V is the command potential, Vrev is the reversal potential (mV), Gmax is the maximal conductance (S), V0.5 is the half-activation potential (mV), and Vslope is the slope factor (mV/e-fold).To examine the voltage-dependent parameters of the sodium current, peak current amplitude from 25 ms test pulse potentials  were measured. Peak IV curves were fitted using: I50 values and neurite outgrowth assays were calculated using a two-way ANOVA followed by a Tukey’s post hoc test. A two-tailed, paired t-test was applied to compare intracellular levels of R123 between resistant and parental cells, as well as CSA-treated and non-treated cells. A two-tailed, paired t-test was also applied to compare current density between retinoic acid differentiated cells to all other experimental groups. All calculations were performed using GraphPad Prism v7.0 . Statistically significant results were represented by p < 0.05 .Statistical analysis of ICN-alkylisatins with varied substituents at the isatin nitrogen retain potency in drug resistant human lymphoma (U937) and uterine sarcoma (MES-SA) MDR cancer cell lines in vitro [N-alkylisatins  and 2ME2-resistant (CEM/2ME2-28.8R) ALL cell lines, and compared to the chemosensitive parental ALL cell line (CEM) using the MTS cell proliferation assay.We have previously demonstrated that 5,7-dibromo-in vitro . In ordeins Fig.  to retai6= 4.869, p < 0.005) decrease in sensitivity to VCR and were significantly cross-resistant to VBL, 2ME2, colchicine and paclitaxel (Table 8 = 4.529 p < 0.05) decrease in drug sensitivity. Interestingly, these cells did not demonstrate cross-resistance to VCR, VBL, colchicine or paclitaxel. Notably, both CEM-VCR R and CEM/2ME2-28.8R resistant cell lines treated with HMB-isatin, I4M-isatin or N2M-isatin showed a negligible change in IC50 values  after 48 h compared to their respective parental cells    differentiated SH-SY5Y cells and measured alterations in neurite length and number of branch points  , indicating a potential for lowered neurotoxicity.To further interrogate neurotoxicity, numbers of neurite branch points were examined following drug treatments Fig. b. At 1 nNeuronal morphology with neuritic-like processes in RA differentiated SH-SY5Y cells is associated with functional features that include transmembrane currents underlying a neuronal excitability phenotype –43. SuchNaïve SH-SY5Y (Ctr) cells displayed relatively small inward currents when compared to RA differentiated cells Fig. a. Treatm0.5: −18.6 ± 0.8 mV), only small depolarizing shifts on the activation midpoint (V0.5) were observed in VBL (V0.5: −13.3 ± 3.2 mV) treated, and naïve (V0.5: −13.8 ± 1.4 mV) cells. The latter also displaying an apparent increase in the slope of the activation curve . When compared to the RA group  and current density  in comparison to vehicle treated control cells , however these changes did not reach statistical significance  and the current density (measured from the ratio of peak current amplitude observed at 0 mV to the cell membrane capacitance in pA/pF) from each group. RA-differentiated SH-SY5Y exposed to HMB-isatin displayed modestly reduced conductance  highlights the need to further develop strategies to treat or prevent ALL relapse. The 5,7-dibromo-N-alkylisatins are a diverse and growing class of antimitotic agents that have demonstrated the ability to retain potency against clinically resistant cancer cell lines in vitro and reduce primary tumour growth in vivo [N-alkylisatins, namely HMB-isatin, I4M-isatin and N2M-isatin, to retain potency against drug resistant ALL cell lines. These N-alkylisatins also demonstrate a potential for reduced neurotoxicity compared to current clinically used Vinca alkaloids, VCR and VBL, suggesting that with further development, the N-alkylisatins could be used in the treatment of ALL with reduced potential for the onset of CIPN.ALL is the most common childhood malignancy and although high remission rates have been achieved, relapse occurs in ~ 20% of patients. This is predominantly due to the development of MDR to chemotherapy . Despite in vivo –30. The N-alkylisatin binding to β-tubulin, as any change in the tertiary binding site and/or its immediate environment may subsequently affect drug activity. In this study, cytotoxicity assays revealed that all three N-alkylisatins retained potency against the CEM/2ME2-28.8R cell line with IC50 values not significantly different to that of the CEM parental control cell line. Our competition binding experiments clearly indicate that N-alkylisatins compete for binding with a colchicine analog, strongly supporting their binding to the colchicine site. Therefore, the fact that the compounds retain potency against the 2ME2  resistaw et al.  and indiVinca alkaloids [50 values were not statistically different to those observed for the parental control CEM cell line. This was in contrast to the dose response profiles observed for the commercial anti-cancer agents and further confirms our findings [N-alkylisatins overcome MDR is yet to be uncovered, but we posit that polypharmacology could play a role [Multiple factors are thought to contribute to the drug resistant phenotype of the CEM-VCR R line , 48. Poilkaloids , howeverlkaloids , 51. LRPlkaloids . Clinicalkaloids . Moreovelkaloids , as welllkaloids . When thfindings  that thiy a role .N-alkylisatins to induce neurotoxicity by comparing their effect on SH-SY5Y neurite length and branch points to VCR and VBL, cytotoxins which are known to cause significant neuronal damage [Vinca alkaloids and N-alkylisatins displayed a dose-dependent effect towards the SH-SY5Y cells, however neurotoxicity associated with VCR and VBL treatment was significantly more pronounced. N-alkylisatins induced less damage to neurites than VCR and VBL, even at 10 × higher concentrations than the Vinca alkaloids. It is important to note that while the potency of VCR and VBL is between 2.7–13-fold greater than the N-alkylisatins against the chemosensitive CEM cell line, the N-alkylisatins were up to 17 × more potent than VCR and 2.3 × more potent than VBL in the CEM-VCR R resistant line, making the concentrations used in the neurotoxicity experiments highly relevant. Of the three N-alkylisatins tested, HMB-isatin was the least neurotoxic, and was therefore chosen for further assessment in functional patch-clamp assays.Antimitotic agents work by causing dynamic instability of microtubules and ultimately induce tumour cell death. They also interfere with the function of microtubules in axons which mediate neuronal vesicle transport –59. For l damage . DiffereFor neurofunctional experiments, we used differentiated SH-SY5Y cells as our model of neurons as they exhibit voltage-gated sodium channels essential to action potential generation and are therefore crucial for neuronal activity and cell survival. The cytotoxic drugs studied here affected sodium current magnitude with varied potency. Macroscopic conductance and current density are influenced by channel expression at the plasma membrane. Our patch-clamp results revealed that the average conductance and current density of HMB-isatin treated cells were not significantly different to those of RA differentiated control cells, which suggests that aspects of channel trafficking and expression in these two groups are akin. In contrast, the sodium currents of VBL and VCR treated cells were significantly smaller and similar in magnitude to the naïve group.v currents, RA and HMB-isatin, fire action potentials upon current injection. VBL treated cells, with somehow lower Nav current, also fired APs but only upon stronger stimuli (i.e. higher current injection), whereas VCR and naïve cells failed to fire under our experimental conditions. Spontaneous firings or trains of spikes in response to prolonged stimulation were not observed in any condition precluding rigorous quantification of the resulting excitability profiles. Perhaps the absence of (repetitive) firing is indicative of incomplete neuronal maturation and/or compromised cell fitness to support firing. Thus, our functional assessment points towards an inherent detrimental role of these drugs in differentiation and/or function of neuron-like SH-SY5Y cells.In our electrophysiological experiments, we observed complete inhibition of SH-SY5Y VGSC currents by 300 nM TTX confirming functional expression of only TTX-sensitive sodium channel isoforms . A detaiv channel behaviour and play critical roles in controlling neuronal excitability [Altogether, morphological and functional indicators from this study suggest that cytotoxic drug treatment affects differentiation towards the neuronal phenotype, as tubulin disrupting drugs, HMB-isatin, VBL and VCR possibly alter channel trafficking and/or expression. Other cytotoxic drugs like platinum compounds, contribute to the development of CIPN by impairing the electrophysiological function of dorsal root ganglion neurons demonstrated by a reduction and/or loss of the sensory action potential in nerve conduction studies . The redtability . Defectitability . Furthertability , 69. Our+ channels. The appearance of large inward TTX-sensitive Nav currents is an electrophysiological marker of neuronal-functionality. As a consequence, RA cells became excitable and thus fired action potentials in response to current injection (Fig. N-alkylisatins within a whole living organism [RA treatment significantly increased the density of the voltage-activated Naion Fig. a, insetsorganism .N-alkylisatin-based compounds with increased anti-cancer activity is ongoing and advancements in the field could lead to novel molecules that possess superior efficacy towards MDR cancers with lessened neurotoxicity profiles. A current opinion on how to best treat ALL relapse is to prevent it, which makes incorporation of these novel drugs into frontline therapy the most practical approach. Combining newer agents with conventional treatment approaches will likely overcome the challenge of balancing efficacy with toxicity and prevent the development of resistant ALL subclones. Altogether, the results obtained from this study reinforce the potential of N-alkylisatins to be considered as candidate drugs for the treatment of patients with relapsed and refractory ALL.The development of Additional file 1: Figure S1. Growth properties of CEM, CEM-VCR R and CEM/2ME2-28.8R cell lines. Figure S2. SH-SY5Y cell viability after treatment with HMB-isatin, VCR and VBL. Figure S3. Dose dependent effect of HMB-isatin and VCR on RA-differentiated SH-SY5Y neurite morphology. Table S1. Electrophysiological parameters of endogenous sodium currents. Table S2. Current density estimates from 3 independent experiments.


Compound 11c showed a broad spectrum in terms of antiproliferative efficacy with GI50 values in the range of 0.296 to 250 μM. Molecular docking studies indicated that Compounds 8a and 11c are accommodated in the colchicine binding site of tubulin in two different ways.Three series of fused pyrrolophenanthroline derivatives were designed as analogues of phenstatin and synthesized in two steps starting with 1,7-phenanthroline, 4,7-phenanthroline and 1,10-phenanthroline, respectively. Two (Compounds  Phenanthrolines are versatile moieties which have found use in many applications ,2,3 due At the same time, the fusion of two or more heterocycle rings can lead to novel classes of ligands, many with new interesting properties, including anticancer activity. For example, substituted benzo[c]phenanthroline analogues of nitidine and fagaronine showed cytotoxic properties associated with DNA intercalation and induced G2/M phases arrests . SynthetDespite the great progress achieved in chemotherapy, cancer remains a leading cause of mortality worldwide ,19. AmonEncouraged by the above considerations  and as part of our ongoing research aimed at investigating new anticancer drugs ,16,28, wWe furthermore considered some modifications at the 3,4,5-trimethoxyphenyl ring of phenstatin (ring A), generating either a 3,5-dimethoxyphenyl, 3,4-dimethoxyphenyl or a 4-bromophenyl ring.In this way, we incorporated the trimethoxyphenyl ring of phenstatin and our fused pyrrolophenanthroline system into a single molecule, in order to investigate their impact on the anticancer activity of the parent compound. Thus, we report here the synthesis and biological evaluation of novel compounds with 1,7-, 4,7-, and 1,10-phenanthroline scaffolds.5a–d were successfully synthesized using a two-step procedure starting from 1,7-phenanthroline 1. The first step comprised the formation of monoquaternary 1,7-phenanthrolin-7-ium salts 3a–d by the nucleophilic substitution of phenanthroline 1 to 2-bromoacetophenones 2a–d .4a–d from the corresponding salts 3a–d under triethylamine treatment. The in situ-formed ylides acted as 1,3-dipoles when reacted with ethyl propiolate, following a Huisgen 3 + 2 cycloaddition. Initially formed unstable intermediates 5′a–d undergo an aromatization process under the current reaction conditions, leading to target compounds 5a–d in 50–88% yields  with ethyl propiolate  were identified by NMR and IR. Compounds ted data ,31,32,335a–d, 8a–d and 11a–d were submitted to the National Cancer Institute  and four compounds were selected for the evaluation of their antiproliferative activity against their panel of 60 human cancer cell lines. The NCI panel is organized into nine sub-panels representing leukemia, lung, colon, central nervous system (CNS), melanoma, ovary, kidney, breast and prostate cancer cells. The panel also includes several multidrug-resistant tumor cell lines . The four compounds—5c, 8a, 8b and 11c—selected by NCI were first tested at a single high dose (10−5 M) in the full 60-cell panel, selected results being presented in 8a and 11c showed very good growth inhibition of several cancer cell lines. The best efficacy in terms of growth inhibition was shown by Compound 11c against the HL-60(TB) RPMI-8226 leukemia cell line, NCI-H522 non-small cell lung cancer line, COLO205 and HT-29 colon cancer cell line, SF-539 CNS cancer cell line, MDA-MB-435 and M14 melanoma cell lines, OVCAR-3 ovarian cancer cell line and A498 renal cancer cell line, these results being comparable or better than phenstatin, which was used as a control. Compound 11c also showed cytotoxic activity against the COLO205, MDA-MB-435 and A498 cell lines. An important growth inhibition effect was also observed for Compound 8a against MDA-MB-435 melanoma cells and K-562, SR and HL-60(TB) leukemia cells. Thus, replacement of ring B of phenstatin with a pyrrolo[c]phenanthroline or a pyrrolophenanthroline group retains the growth inhibition properties of phenstatin. The more active Compound 8a, by comparison with 8b, retains the three methoxy groups of the more active phenstatin. Therefore, the lack in the methoxy substituent in para position of the ring B appears to be not favorable for the anticancer activity, at least in the case of compounds type 8.Compounds lophenanthr11c, which had the best growth inhibition profile among the tested compounds, progressed to the full five-dose assay. Selected GI50 values are presented in 11c possess excellent to moderate antiproliferative activity with GI50 values ranging from 0.296 to 3.78 μM against 40 cancer cell lines from all nine sub-panels. In particular, Compound 11c showed the best GI50 and TGI  values (296 nM and 981 nM respectively) against MDA-MB-435 melanoma cells. Promising GI50 values were also obtained for against NCI-H522 lung cancer cells, HCT-116 colon cancer cells and M14 melanoma cells. However, when comparing to control phenstatin, there are only a few cell lines against which Compound 11c is more potent  or shows similar antiproliferative activity .Compound 8a and 11c and the lack of activity in the case of 5c and 8b.In order to verify if our target phenstatin analogues retain the ability of their parent compound to fit in the colchicine binding site of tubulin, docking studies were performed on the four NCI-tested compounds in the colchicine binding site of the α,β-tubulin heterodimer (PDB:1SA0). The in silico study aimed to evaluate the shape and electrostatic complementarity between the tested ligands and the α,β-tubulin heterodimer interface, which could account for the observed antiproliferative effects in the case of 8a and 8b in the colchicine binding site of tubulin revealed a similar accommodation to previously reported ligands phenanthroline group appears to be the most beneficial, while the replacement with a pyrrolo[8 and 11 are amenable for further structural optimization in the development of chemotherapeutic agents.Three new classes of potential phenstatin analogues have been synthesized and characterized. Four compounds were selected by NCI and tested against a panel of 60 cell lines at a single concentration of 10


Ferula L., belonging to the Apiaceae family, dicotyledonous plants present in many temperate zones of our planet. Ferula communis L. is the main source of sesquiterpene ferutinin, a bioactive compound studied both in vitro and in vivo, because of different effects, such as phytoestrogenic, antioxidant, anti-inflammatory, but also antiproliferative and cytotoxic activity, performed in a dose-dependent and cell-dependent way. The present review will focus on the molecular mechanisms involved in the different activities of Ferutinin, starting from its antioxidant potential at low doses until its ionophoric property and the subsequent mitochondrial dysfunction induced through administration of high doses, which represent the key point of its anticancer action. Furthermore, we will summarize the data acquired from some experimental studies on different cell types and on several diseases. The results obtained showed an important antioxidant and phytoestrogenic regulation with lack of typical side effects related to estrogenic therapy. The preferential cell death induction for tumor cell lines suggests that ferutinin may have anti-neoplastic properties, and may be used as an antiproliferative and cytotoxic agent in an estrogen dependent and independent manner. Nevertheless, more data are needed to clearly understand the effect of ferutinin in animals before using it as a phytoestrogen or anticancer drug.The employment studies of natural extracts in the prevention and treatment of several diseases highlighted the role of different species of genus  The use of herbal and plants extracts to prevent and to treat several acute and chronic diseases is an object of growing interest and investigation in pharmacological and nutraceutical research ,2,3.In vitro and in vivo evidence showed that the bioactive compounds isolated from herbs and plants showed antioxidant, anti-inflammatory and anti-cancer property ,5,6. The oxidative-stress reduction leads to a decreased risk of onset of several pathologies, such as cancer and chronic inflammatory diseases . Among tThe results obtained in different experimental works suggest that the sesquiterpenes have protective potential at low doses and cause severe toxic effects at high doses ; this biFerula L. is one of the most representative .,83.82,83Increasing evidence on the ability of ferutinin to inhibit cell growth and to induce apoptosis both in vitro and in vivo  has led In addition, the SMAC/DIABLO molecule, inhibitor of the inhibitors of apoptosis proteins (IAPs), might be involved in the mechanism of ferutinin toxicity, as once leaked from the mitochondria, it can bind IAP, preventing the inhibition of caspase activity and promoting the formation and activation of the apoptosome complex, leading to caspase-9 activation ,91 that Among cell lines tested to verify ferutinin effects, it was observed that, for tumor cell lines, human breast adenocarcinoma cell line (MCF-7), human cells of urothelial carcinoma (TCC), human colon adenocarcinoma cells (HT29), murine cells of colon carcinoma (CT26), IC 50 was between 67 and 81 μM, while the IC 50 for normal human and murine fibroblast lines (HFF3 and NIH/3T3) was higher, with values of 98 and 136 μM respectively, with about 67% of viable cells in both cell lines treated at the above concentrations  32,33,3,35,36. AFurthermore, Arghiani et al. (2015) observed cytotoxic effects of ferutinin on human teratocarcinoma (NTERA2) and oesophageal cancer (KYSE30) cell lines and the IC50 values were respectively 39 μM and 58 μM .The anti-cancer ferutinin activity was also evaluated in vivo, in comparison with cisplatin, on tumors developed in hybrid BALB/C mice: a tumor reduction of 67% for ferutinin and of 72% for cisplatin was observed; however while the latter produced a significant alteration of the liver tissues and the spleen of the treated mice, this did not happen for mice in treatment with ferutinin . In vitro studies have shown that, thanks to its phytoestrogenic activity, ferutinin induced the inhibition of proliferation in human breast carcinoma MCF-7 cells . To testA recent study confirmed the cytotoxic activity of ferutinin also on the prostate cancer (PC-3) cell-line, with IC 50 values of 16.7 µM. The low IC 50 is due to the cross-link among the cytotoxic activity and hormonal action, since the prostate cancer onset is primarily due to androgenic hormones .Gao et al. (2013) showed that ferutinin, in a dose-dependent way and, particularly, at high concentrations (40 μM or above), was able to induce apoptosis in human red blood cells (RBCs), without the involvement of mitochondria since these cells lose those organells during the maturation process. The erythroptosis is mediated by simultaneous increase of the cytosolic free calcium ions level and the activity of caspase-3. In addition, the external calcium depletion had not blocked the apoptosis mediated by ferutinin, suggesting that the existence of unknown mediators of apoptosis are ferutinin-induced, which could be added to the ionophoric activity . CertainFerula genus extract contains a high percentage of the bioactive compound ferutinin, having a wide range of biological effects that elect it as a possible therapeutic agent with a broad spectrum of action. We examined the effects of ferutinin, highlighting the deep gap existing between the uses of high and low doses.The Indeed, the ferutinin administration at low-concentration is associated with an important antioxidant action, although the mechanisms of free radical decrease still need further clarification.Moreover, the effects of cross-talk between the estrogenic and ionophoric properties of ferutinin that lead to strongly reduction of the side effects produced by usual estrogen therapy, allow this molecule to be eligible as a potential therapeutic agent in the gynecological pathologies. Concerning the antiproliferative action of ferutinin, several in vitro and in vivo studies showed that the bioactive compound acts both on cytotoxicity and cell proliferation in an estrogen-dependent and -independent manner and its activity is dose dependent and variable according to the cell type analysed, with a preferential action for tumor cell lines.The ionophoric effect of ferutinin with the consequent loss of the trans-membrane potential seems to be the key point of the antiproliferative and cytotoxic effect, both directly through the activation of mitochondrial-dependent apoptosis and indirectly through the effects produced by the summarization of NO and ROS, molecules that play a key role in the production of stress, senescence and death signals.Therefore, based on the evidences obtained, the bioactive compound ferutinin can be considered an effective therapeutic agent as adjuvant or substitute for the current anti-neoplastic treatments. More studies should be performed to evaluate its action on other cell types and further molecular pathways involved, to compare its activity with that of chemotherapy drugs going to evaluate the different degree of toxicity produced as a side effect on healthy cells.


A recent genome-wide association study (GWAS) of 59 cerebrospinal fluid (CSF) proteins with a connection to Alzheimer’s disease (AD) demonstrated an association between increased levels of chemokine ligand 2 (CCL2) with an atypical chemokine receptor chemokine-binding protein 2 variant V41A . High levels of CCL2 are associated with increased risk of AD development as well as other inflammatory diseases. In this study we characterized the biological function of the ACKR2-V41A receptor compared to the wild type allele by measuring its ligand binding affinity, CCL2 scavenging efficiency, and cell activation sensitivity. We transfected Chinese hamster ovary cells with plasmids carrying wild type ACKR2 (ACKR2-WT) or the mutant ACKR2-V41A receptor. Binding affinity assays showed that ACKR2-V41A has a lower binding affinity for CCL2 and CCL4 than ACKR2-WT. CCL2 scavenging results aligned with binding affinity assays, with ACKR2-V41A cells scavenging CCL2 with a lower efficiency than ACKR2-WT. Cell activation assays also showed that ACKR2-V41A cells had significantly lower receptor upregulation  upon stimulation compared to ACKR2-WT cells. These findings provide molecular and biological mechanistic insights into the GWAS association of ACKR2-V41A with increased levels of CCL2 in CSF and possibly other chemokine ligands. Increased CCL2 levels are associated with accelerated cognitive decline and increased risk of AD. Understanding how this atypical chemokine receptor allele increases serum markers of inflammation could lead to novel therapeutic solutions for AD. Acute inflammation plays an essential role in the normal response to tissue injury3. This inflammatory response initiates a cascade of cellular activation signals in innate immune cells , resulting in increased production of proinflammatory cytokines and chemokines6. These cytokines and chemokines are essential to the recruitment and activation of other cells in the innate and adaptive immune systems. An inappropriate response to inflammation or alterations in the production of these chemokines can result in disease development.Inflammation is associated with increased risk of developing cancer, autoimmune disorders, and neurodegenerative diseases such as Alzheimer’s disease (AD)7. Significant progress has been made towards understanding the genetic architecture of AD. To date, 32 loci have been identified with common and rare alleles that influence the risk of AD10. AD is pathologically characterized by the accumulation of amyloid beta (Aβ) peptide forming insoluble aggregated plaques in the gray matter and is thought to result in neurodegeneration and loss of memory function11. However, the characterization of biological mechanisms by which these rare variants and the accumulation of Aβ modulate risk for disease has proven to be extremely difficult, with only several of these variants being experimentally characterized19.AD is the most common neurodegenerative disorder and is the main cause of dementia in the elderly population20. This genome-wide association study (GWAS) of 59 AD-related CSF analytes used two independent datasets: the Knight Alzheimer’s Disease Research Center (ADRC) and Alzheimer’s Disease Neuroimaging Initiative (ADNI)20. This study demonstrated an association between increased levels of chemokine ligand 2 (CCL2) with atypical chemokine receptor chemokine-binding protein 2 (ACKR2) variant V41A.Recent analyses of quantitative AD endophenotypes in cerebrospinal fluid (CSF) has identified a chemokine receptor variant that alters the risk and rate of progression of ADCCL2 gene located on chromosome 17q11.2-q12. CCL2 is a pro-inflammatory chemokine involved in recruitment of immune cells from the blood to sites of inflammation via chemokine gradients. CCL2 helps to control blood brain barrier migration of monocytes and dendritic cells and aids in the differentiation and migration of macrophages21. In the brain, CCL2 is mainly secreted by astrocytes, microglia, and in low levels by endothelial cells22. Upon an immune response to infection, injury or inflammation, CCL2 is produced in the central nervous system (CNS)23. CCL2 interacts with chemokine receptor CCR2 which signals via a G couple protein cascade. Upon CCR2 and CCL2 ligand interaction, a cascade of cell activation events takes place . This activation cascade signals cell migration, cell survival, transcription regulation, and release of pro-nociceptive molecules25. Upon CCL2 binding to a receptor, it induces a strong chemotactic response and mobilization of intracellular calcium ions and synaptic network activity in the hippocampal neurons27. High levels of CCL2 is a risk factor in several neuroinflammatory and neurodegenerative brain diseases such as multiple sclerosis, brain ischemia, traumatic brain damage, and AD28. In AD mouse models, CCL2 is key to inducing chronic inflammation and activation of immune cells, and secretion of other chemokines29. Overexpression of CCL2 retains activated microglia cells near the inflamed site30. This results in increased interactions of activated microglia with key features of AD: amyloid plaques, plague aggregation, and cognitive decline33. Inhibition or removal of CCL2 in AD mouse models revealed accelerated amyloid pathology formation34. These studies demonstrate that proper function and regulation of CCL2 is imperative to preserve brain innate immune response homeostasis and cognitive function.CCL2 is also known as monocyte chemotactic protein-1 (MCP-1) and is encoded by the 35) is encoded by the ACKR2 gene located on chromosome 3p21.3. ACKR2 is a seven transmembrane G-protein-coupled receptor containing three intracellular loops and three extracellular loops36. ACKR2 binds with the 14 inflammatory chemokines of the CC family, including CCL2 and CCL4, but not CCL1937. ACKR2 is an atypical chemokine due to its lack of a canonical DRYLAIV motif in the second extracellular loop. This lack of motif inhibits the ability of the receptor to signal the production of other cytokines36. Instead, ACKR2 recognizes inflammatory chemokines, scavenges them, and upregulates the production of more ACKR2 receptor38. ACKR2 plays an essential role in the regulation of the inflammatory response by internalizing inflammatory chemokines, facilitating their destruction when the cellular endosome fuses with an acidic lysosome. The chemokine-free ACKR2 receptor is then recycled back to the cell surface and can scavenge additional inflammatory chemokines39. ACKR2 is mainly expressed on leukocytes, including dendritic cells, monocytes, macrophages, and innate-like B cells40. In the central nervous system (CNS) ACKR2 is mainly expressed in microglia and astrocyte cells43. Nonsynonymous mutations mapped to the ACKR2 gene have been associated with increased risk for breast cancer, testicular leukemia, CD45 deficiency, glucose intolerance and inhibit leukocyte type differentiations and monocyte counts49.Atypical chemokine receptor 2 , rs2228467 was associated with increased coronary heart disease and a higher monocyte count in the blood stream51. Rs2228467 was also identified as an increased risk factor for developmental disorders following the analysis of over 700 mother-infant pair mid-gestational serum and neonatal bloodspots52. Another 2017 study using the National Human Genome Research Institution (NHGRI) dataset found that rs2228467 is associated with increased AD risk53. Together these studies make a compelling case for the biological characterization of ACKR2-V41A. Here we provide the molecular and biological characterization of ACKR2-V41A and show that it has decreased binding affinity, scavenging efficiency, and receptor upregulation  for CCL2. These results provide insights into the previously reported association of ACKR2-V41A with AD risk, rate of cognitive decline, and serum markers of inflammation which could lead to novel therapeutic solutions for AD.The ACKR2-V41A allele (rs2228467) is associated with altered chemokine levels in the CSF and bloodstream. A 2017 study with 8,293 Finn participants identified increased levels of the chemokine eotaxin (a potent eosinophil attractant) associated with rs2228467 and increased risk of developing Crohn’s disease, multiple sclerosis, and ulcerative colitisACKR2-V41A differs from wild type by a single amino acid located at the chemokine binding site of ACKR2. V41A is specifically located in the first of three extracellular loops. Predicted protein folding models were generated and ACKR2-WT and ACKR2-V41A were superimposed to check for differences in folding and hydrophilicity. There was no observed difference in predicted protein structure folding Fig. .Figure 1RegulomeDB gave this nonsynonymous mutation a score of 5/6. PolyPhen-2 predicted this variant to be probably damaging with a score of 0.958 . These predictor tools - Chimera, RegulomeDB and PolyPhen-2 all predicted that the ACKR2-V41A variant causes a biological change with functional consequences.The single amino acid change at position 41 is predicted to have a slight decreased hydrophilicity between ACKR2-WT and ACKR2-V41A Fig. . DespiteCHO-k1 cells were transfected with ACKR2-WT or ACKR2-V41A and were grown in selective media for a week before transfection positive cells were enriched using beads and magnetic columns. The enriched CHO-k1 ACKR2-WT or ACKR2-V41A cell populations had similar percentages of receptor positive cells Fig.  and receThe binding affinity of ACKR2-WT and ACKR2-V41A for CCL2 was measured in both a direct binding and a competition assay. In the direct binding assay, the percentage of CCL2 binding to ACKR2-WT and ACKR2-V41A was determined by measuring the levels of AF647-conjugated CCL2 divided by the ACKR2 receptor levels. Analysis of 5 independent runs revealed that ACKR2-V41A has a significantly lower binding affinity than ACKR2-WT Fig. . Non-linA competition assay was also performed to test if the unconjugated chemokine ligand CCL2 was able to interfere with binding of an anti-ACKR2 antibody. Analysis of 5 independent runs showed a significant difference between chemokine receptor ACKR2-WT and ACKR2-V41A . ACKR2-V41A expressing cells bound to significantly lower levels of CCL2, allowing the ACKR2 antibody to bind at a higher percentage (μ=88.5) than ACKR2-WT (μ = 66.18). Quantification of levels of unconjugated CCL2 blocking the anti-ACKR2 antibody show that ACKR2-V41A (11.5%) binds with significantly lower affinity to CCL2 than ACKR2-WT (33.82%)  than ACKR2-WT (26.0%) Fig. . Under t50 of 18.19 hours, illustrating that the WT receptor scavenges CCL2 more efficiently than ACKR2-V41A, which has an IC50 of 36.83 hours  induced by CCL2. Upon activation of the receptors with chemokine ligand CCL2, cells expressing ACKR2-V41A receptor had significantly lower p-cofilin levels than cells expressing ACKR2-WT  imaging. Upon activating the receptor with chemokine CCL2, cells expressing the ACKR2-V41A receptor have significantly lower levels of calcium signaling starting 4 minutes after CCL2 activation when compared to CCBP-WT  and a possible mechanism for increased levels of CCL2 in the CSF. ACKR2-V41A has lower CCL2 binding affinity, CCL2 scavenging efficiency, and CCL2 induced ACKR2 receptor upregulation. As proposed in the original GWAS analysisChimera protein predictor software, PolyPhen-2, and Regulomedb predicted that the amino acid change from the hydrophobic valine to the less hydrophobic alanine was sufficient to change biological function. In this study, we demonstrated that variant V41A, located at the first extracellular loop and binding site, reduces the binding affinity of receptor ACKR2 with CCL2 and CCL4 ligands. We suspect that same behavior will be observed if the binding affinity of this variant is tested with the remaining 12 chemokine ligands known to interact with it. In addition, ACKR2-WT and ACKR2-V41A did not noticeably improve binding for the chemokine ligand CCL19. This ligand is one of the chemokines from the CC family that does not interact with ACKR2 receptor. Thus, in this case the V41A variant did not enable the ACKR2 receptor to interact with a new chemokine ligand.This altered ability of ACKR2 to bind CCL2 resulted in decreased CCL2 scavenging activity and lower levels of receptor on the cell surface. ACKR2-V41A lost its scavenging efficiency over time (after 18 hours of saturating the system with 100 pg/ml of CCL2) and also has decreased levels of the ACKR2 receptor at the cell surface over time (starting at 18 hours after the cells were stimulated with 100 pg/ml of CCL2) compared to wild type. We found that ACKR2-V41A is two-fold less efficient at scavenging CCL2, resulting in the maintenance of this inflammatory chemokine signal for a longer time than wild type. These results suggest that binding affinity and receptor levels are key to regulating levels of pro-inflammatory chemokines such as CCL2.55. Here we report that after CCL2 activation, p-cofilin expression levels were reduced in cells with the ACKR2-V41A receptor compared to wild type. This result aligns with the assay examining ACKR2 receptor levels after CCL2 stimulation over a 72 hours period. Low levels of p-cofilin in ACKR2-V41A cells could explain the low level of ACKR2 quantified at the cell surface and the decreased CCL2 scavenging activity. In addition, there are compelling studies showing that ACKR2 is capable of Ca2+ mobilization upon stimulation with CCL2 in in HEK 293 cells56 and neurons57. These studies align with our results and strengthen our proposed mechanism. Here we have shown a reduction of the biological capacity of the atypical chemokine ACKR2-V41A to bind, scavenge, and be activated by CCL2. Additional impacts of the ACKR2-V41A allele should also be examined in regard to the other 12 chemokines ACKR2 is known to bind with37. Thus, alterations in chemokine levels caused by ACKR2-V41A have a powerful effect on chemokine gradient homeostasis, inflammation, and increased risk to develop AD.ACKR2 is an atypical chemokine receptor and lacks the signaling motif to initiate the production of other chemokine and cell migration. ACKR2 stimulation by CCL2 activates the β-Arrestin-dependent signaling pathway cofilin pathway [Rac1-p21-activated kinase 1 (PACK1)-LIM kinase 1 (LIMK1) cascade] and ACKR2 can send an intracellular signal to upregulate surface receptor levels59. Thus, regulation of CCL2 levels has multiple effects on brain homeostasis and longevity. In this study, we observed significant differences in the ability of ACKR2-V41A to bind and scavenge CCL2, potentially resulting in higher levels of CCL2 at the injury site and maintenance of pro-inflammatory signaling and monocyte recruitment, which may lead to increased Aβ plaque aggregation and risk for AD.In the presence of Aβ aggregation the expression and production of pro-inflammatory related molecules, including CCL2, are elevated. In this context, it is known that high levels of CCL2 expression and production trigger microgliosis, increase the APP/CCL2 ratio, and increase Aβ plaque aggregation. CCL2 could increase the rate of Aβ deposition by interference with clearance as well as increase expression of apolipoprotein E, a main biomarker for AD pathogenesis and Aβ deposition60. Consequently, genetic variants identified by GWAS have to be physiologically significant to conclusively identify their biological effects as proteins. In this study we provide the biological characterization of ACKR2-V41A that was identified using a GWAS. We have provided insights into the mechanism by which ACKR2-V41A with CCL2 alters the chemokine gradient homeostasis, increases inflammation, and the alters risk for AD. Future efforts should focus understanding if and how ACKR2-V41A impacts Aβ production and aggregation and other biomarkers of AD in AD-relevant cell lines.GWAS researchers have identified thousands of genetic variants contributing to the rate of development of complex human diseases; providing insights at the DNA level of how a single nucleotide polymorphism (SNP) might cause changes in function at the biological and molecular level. The characterization of biological mechanisms by which rare variants found in GWAS to modulate risk for disease has proven to be extremely difficult. SNP characterization requires a deeper understanding of how DNA changes might affect gene expression through transcription, RNA splicing, ligand-receptor binding, protein function etc62. These 11 models were compared to similar chemokine receptors for accuracy. Using UCSF Chimera Software, the 3D models were oriented in space63. We superimposed ACKR2-WT and ACKR2-V41A to check for differences in folding and hydrophilicity. 3D images were produced using UCSF Chimera Software to highlight the predicted chemokine receptor structure and hydrophobic interactions at amino acid position 4163.ACKR2-WT and ACKR2-V41A chemokine receptor amino acids sequences were sent in a FASTA format to the University of Michigan’s Zhang Lab LOMETS server. 3D models were generated by collecting high-scoring structural templates from 11 locally-installed threading programs 64. This predicter algorithm scores between 1 and 6 and any variant categorized between 1 and 5 is predicted to have damaging biological function in the human genome. PolyPhen-2 is an algorithm that predicts possible biological impact of an amino acid substitution in the human genome65. This algorithm classifies the biological impact of a variant from a damaging to a tolerable variant.Annotation and prediction of the biological function of ACKR2-V41A variant was determined using RegulomeDB and Polymorphims Phennotyping v2 (PolyPhen-2). RegulomeDB is a database that annotates variants with known and predicted regulatory elements in human genome such as eQTL, transcription factor binding and transcription factor motifa cells, amplified, and purified using the ZymoPURE II Plasmid Maxiprep kit (Cat #D4203). Plasmids were transfected into Chinese Hamster Ovary (CHO-k1 ATCC® CCL-61) cells using lipofectamine  and manufacture recommendations were followed. Properly transfected cells were selected using the antibiotic G418 sulfate . Transfected cells were grown in F12 media  with 10% FBS (HYCLone Cat #SH30071.01). Cell media was changed every 48 to 72 hours depending on cell confluency levels.Plasmid construct were ordered at Genscript. DNA sequences for ACKR2-WT and ACKR2-V41A were inserted into vector pCMV6-AN-myc-DDK . Transformation protocols were followed as recommended by the manufacturer. Briefly, plasmids were transformed into DH5We tested the atypical chemokine receptor ACKR2-V41A function using CHO-k1 cells because they lack expression of the wild type ACKR2 receptor as well as other competing human chemokine receptors. It is important to note that this cell type is absent in the human brain and therefore our results should not be overinterpreted. After transfecting CHO-k1 cells with plasmids carrying ACKR2-WT or ACKR2-V41A, cells were incubated until they reached 80% confluency and prepared for cell sorting. Cells were stained for 30 minutes at 37 °C with anti-human ACKR2 polyclonal IgG antibodies (ABNOVA Cat #H00001238-B02). After two washes, cells were stained for 30 minutes at 4 °C with anti-human IgG conjugated with PE (Biolegend Cat #405307). Cells were then washed and isolated using anti-PE MicroBeads from MACS Miltenyi Biotech (Cat #130-048-801) following manufacture recommendations. After isolation, cells were incubated in F12 media until 80% confluency was reached.2 flask reached 80% cell confluency. 250,000 cells were resuspended in 300 μL of PBS-1% BSA and placed in FACs tubes. To evaluate expression of the ACKR2 receptor, cells were stained with anti-CD16/32 (Fc block) on ice for 20 minutes . After two washes, cells were stained for 30 minutes at 37 °C with anti-human ACKR2 polyclonal IgG antibodies, washed twice, and then stained for 30 minutes at 4 °C with anti-human IgG conjugated with PE. After two washes receptor expression was analyzed on a BD Accuri. In addition, expression of the receptor at cell surface was monitor for 72 hours after CCL2 was added to the growing media. Expression levels were obtained following the procedures above.CHO-k1 cells transfected with ACKR2-WT or ACKR2-V41A were harvested when a 75 cmTo determine CCL2 binding levels for ACKR2-WT and ACKR2-V41A, cells were incubated with conjugated CCL2-AF647  at 37 °C for 45 minutes at different concentrations ranging between 100 to 750 nM. After two washes receptor expression was analyzed on a BD Accuri. To determine the levels of ACKR2-WT and ACKR2-V41A receptor expression, cells were stained with anti-CD16/32 (Fc block) on ice for 20 minutes. After two washes, cells were stained for 30 minutes at 37 °C with anti-human ACKR2 polyclonal IgG antibodies, washed twice, and then stained for 30 minutes at 4 °C with anti-human IgG conjugated with PE. In the CCL2 competitive binding assay levels for ACKR2-WT and ACKR2-V41A, cells were incubated with unconjugated CCL2  at 37 °C for 45 minutes. After two washes and an Fc block as described above, cells were stained for 30 minutes at 37 °C with anti-human ACKR2 polyclonal IgG antibodies, washed twice, and then stained for 30 minutes at 4 °C with anti-human IgG conjugated with PE. After two washes, levels of binding of the ACKR2 antibody was analyzed in BD Accuri.CCL4  competition binding assay was performed following the procedures above. Conjugated CCL19-AF647  binding analyses were completed using the same methods used for CCL2 binding assay.ACKR2-WT and ACKR2-V41A cells were seeded in a 6-well cell culture plate with fresh F12 media with 10% FBS and 100 pg/mL of CCL2 added to each well. At varying time points, 150 μL of media supernatant was extracted from each well to measure the remaining concentration of CCL2 using a CCL2 TMP sandwich ELISA kit . Ninty-six well ELISA microplates were coated overnight at room temperature with a monoclonal antibody specific for human CCL2 (0.25 μg/mL in PBS). Plates were washed three time with 0.05% tween diluted in PBS and then wells incubated with blocking buffer (PBS/1% BSA) for 2 hours at room temperature. After three washes, samples and controls were added to the wells and incubated for 2 hours at room temperature. After three washes plates were incubated for 1 hour at room temperature with biotinylated anti-human CCL2 antibody (0.25 μg/mL) diluted in blocking buffer. After three washes, plates were incubated with 100 μL of Streptavidin-HRP conjugate (0.05 μg/mL) for 30 minutes at room temperature. Plates were washed three times and 100 μL of TMB substrate solution (hydroperoxide and chromogen tetramethylbenzidine) was added for 20 minutes at room temperature in the dark. The reaction was stopped with 100 μL of 1 M of hydrochloric acid. Optical density was determined using a micro plate reader set at 450 nm with a wavelength correction of 620 nm.55. Briefly, 300,000 CHO-k1 cells expressing either ACKR2-WT or ACKR2-V41A were seeded in a well and serum-starved for 18 hours. Then, cells were stimulated with 100 nM of CCL2  and incubated at 37 °C in 5% CO2 for 30 minutes. Cells were harvest, lysed using RIPA lysing buffer  following manufacture recommendations. Lysed product was prepared for a western blot assay. Two gels were run in parallel using the same lysed product samples. One gel was stained with antibody for p-cofilin  to detect phosphorylated cofilin. ImageJ  was used to analyze the intensity of bands corresponding to p-cofilin. The second gel was stained with cofilin  and was used for normalization of p-cofilin after ImageJ analysis. An independent T test was performed to determine if p-cofilin levels differ between cell expressing ACKR2-WT and ACKR2-V41A.Levels of phosphorylated cofilin were obtained following methodology from previously described research2. The next day, cells were loaded with 4 μM of Fura-2AM  in Ringers solution  for thirty minutes at 37 °C in 5% CO2. Cells were then washed once with Ringers solution and incubated for another 30 minutes at 37 °C in Ringers solution. Calcium imaging was performed at room temperature using an Olympus IX51 inverted microscope equipped with a xenon arc lamp. Fura-2AM loaded CHO-k1 cells expressing receptor ACKR2-WT and ACKR2-V41A were excited using 340 nm and 380 nm excitation filters, and images of 340 nm, 380 nm, and transmitted light was captured using a fluorescence microscope camera (Q Imaging Exi Blue) with a 20x objective (N.A. 0.75) at 1.6-sec intervals. At the one minute time point in each imaging protocol, 250 nM of recombinant Human CCL2 was added to stimulate Ca2+ flux23. Ionomycin  was added at the five minute time point as a positive control. Ten to twenty representative cells were selected as regions of interest in each frame, and F340:F380 ratios were calculated and analyzed using CellSens software from Olympus. Each individual cell’s fluorescence was normalized to its first recorded value according to the equation (F-Fo)/Fo, where F is the fluorescence at the specific time point, and Fo is the fluorescence value at time 067.Cells were seeded on a 8-chambered slide  and incubated overnight in F12 media with 10% FBS at 37 °C in 5% CO50, and IC50 were calculated using non-linear analysis best fit test. Each independent run contained a minimum of three replicates per sample and the mean of those replicates were considered a single point of analysis. In this study, each experiment was performed with three to five independent runs (9 to 15 replicates per sample). Results presented are representative of observed phenotypes and analysis with P-values less than 0.05 were considered significant.Statistical analyses were performed using the Prism 8 software . Differences in binding affinity, CCL2 scavenging efficiency, and cell activation sensitivity between ACKR2-V41A and ACKR2-WT were analyzed using an unpaired T-test. Kd, ECSupplementary information.


Natural extracts, including nobiletin, have been reported to enhance the efficacy and sensitivity of chemotherapeutic drugs. However, whether and how nobiletin affects tumor growth and progression in renal cell carcinoma (RCC) are still unclear.in vitro. The nude mouse model was used to test the efficacy of nobiletin alone or in combination with palbociclib.Cell proliferation, cell cycle and apoptosis analyses, colony-formation assays, immunoblotting analysis, and qRT-PCR analysis were performed to investigate how nobiletin affected RCC cell proliferation in vitro and in an in vivo model.Nobiletin inhibited cell proliferation by inducing G1 cell cycle arrest and cell apoptosis in RCC cells. Mechanistically, nobiletin decreased SKP2 protein expression by reducing its transcriptional level. The downregulated SKP2 caused accumulation of its substrates, p27 and p21, which further inhibited the activity of the G1 phase-related protein, CDK2, leading to inhibition of cell proliferation and tumor formation. A higher SKP2 protein level indicated less sensitivity to the CDK4/6 inhibitor, palbociclib. A combination of nobiletin and palbociclib showed a synergistic tumor inhibition Nobiletin downregulated the SKP2-p21/p27-CDK2 axis to inhibit tumor progression and showed synergistic tumor inhibition effects with the CDK4/6 inhibitor, palbociclib, on RCC, which indicates a potential new therapeutic strategy. Despite a higher proportion of indolent and localized tumors identified partly due to widespread use of abdominal imaging techniques, there are still up to 17% of patients with distant metastases at the time of diagnosis2. In addition, approximately 25% of patients with localized RCC develop distant metastasis after radical surgery. Once progressed to metastatic RCC (mRCC), the treatment is still an intractable problem, despite the introduction of novel agents targeting different pathways including angiogenesis, mTOR inhibitors, and immune checkpoint inhibitors. Compared with cytokine treatment , targeted therapies have significantly improved the clinical outcomes of mRCC3. Sunitinib, as a first-line therapy for RCC, achieved significantly longer progression-free survival (11 vs. 5 months), overall survival (OS) (26.4 vs. 21.8 months), and a better response percentage (31% vs. 6%) than the interferon alpha group5. However, due to the intratumor and intertumor heterogeneities of RCC, the objective response percentages of current first-line agents are approximately 30% or lower6. Moreover, almost all patients eventually suffer drug resistance, which suggests that new targets and treatment strategies need to be identified to effectively treat these patients.Renal cell carcinoma (RCC) is the 6th most common cancer in men and the 10th in women, accounting for 5% and 3% of all new cancer cases, respectively8. Some extracts have anti-tumor pharmacological effects and can be used as a regulator of multi-drug resistance, to increase the sensitivity of cancer cells to chemotherapy9. Nobiletin is a typical example, which is an O-methylated flavonoid found mainly in citrus peel. Previous studies have shown that nobiletin significantly inhibited tumor cell growth and metastasis both in vitro and in vivo through multiple pathways11. Nobiletin can also significantly inhibit cell proliferation by attenuating the expression of cyclin D1, CKD2, CKD4, and E2F12. Nobiletin inhibits RCC cell viability and hypoxia-induced migration, which might be induced by the SRC/AKT, NF-κB, and Wnt/β-catenin signaling pathways14. Although nobiletin has been reported to induce G0/G1 cell cycle arrest in RCC cells, the mechanism of action is still unclear14.Many natural extracts are potential antitumor drugs because of their low toxicity and few side effects15. G1/S cell cycle transition is mainly regulated by two cyclin/CDK complexes, cyclinD-CDK4/6 and cyclinE-CDK2, which have been reported to trigger G1/S transition by promoting RB (retinoblastoma) phosphorylation16. Abnormal CCND-CDK4/6-INK4-RB signaling has been involved in tumorigenesis and in tumors21, and represents a valid therapeutic target16. Palbociclib is an orally active, potent, and selective inhibitor of CDK4 and CDK6, which blocks RB phosphorylation at low drug concentrations22. Palbociclib can inhibit RCC cell proliferation at nanomolar concentrations23, and has been tried in phase II studies to treat various types of solid tumors26. However, the effects of CDK4/6 inhibitors have been limited by the other bypass signal27. Some studies have shown that palbociclib blocked cells in the G1 phase by inhibiting CDK4/6 activity, but it did not inhibit CDK2 activity, and the inhibitory effect of RB phosphorylation could be reversed by CDK2 to produce drug resistance28, which indicated that a combination CDK2 inhibitor with palbociclib may increase the therapeutic effect.Cell-cycle dysregulation is prevalent in multiple malignancies, including RCCIn this study, we found that nobiletin induced G1-phase cell cycle arrest, apoptosis, and proliferative inhibition in RCC cells. Nobiletin downregulated mRNA expression of SKP2 by upregulating the transcription factor, FOXO3A, leading to accumulation of p21 and p27 and the reduction of CDK2, to inhibit cell proliferation and tumor formation. We further found that the sensitivity of the CDK4/6 inhibitor, palbociclib, was negatively associated with SKP2 protein levels. Furthermore, a combination of nobiletin and palbociclib showed synergistic lethality, which suggests this combined regimen may be a therapeutic strategy for RCC.2. All cell lines were tested and were shown to be free of mycoplasma contamination.Nobiletin, palbociclib, MG-132, and cycloheximide were purchased from Selleck Chemicals , and dissolved in dimethyl sulfoxide (DMSO)  and stored at -20 °C. The RCC lines  and the immortalized epithelial renal cell line (HK-2) were purchased from the Zhong Qiao Xin Zhou Biotechnology . The 786-O, 769-P, and OSRC-2 cell lines were routinely maintained in RPMI-1640 medium. The Caki-1 cell line was cultured in McCoy’s 5A medium, and the HK-2 cell line was cultured in KFSM medium. All media were supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin except KFSM medium. All media were purchased from Gibco . The cells were cultured at 37 °C in a humidified atmosphere containing 5% COFor direct IB analysis, the cells were lysed in radioimmunoprecipitation assay (RIPA) buffer with phosphatase inhibitors. The following primary antibodies were used against: cleaved caspase-3 (Asp175) (5A1E) (#9664), cleaved caspase-8 (Asp391) (18C8) (#9496), RB (#9309), p-RB (Ser870/811) (#8516), CDK2 (#2546), p-CDK2 (#2561), Bcl-2 (#15071), Bax (#5023), p21 (#2947), p27 (#3686), cyclin D1 (#55506), cyclin E (#4136), p44/42 MAPK (Erk1/2) (137F5) (#4695), phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) XP (#4370), Akt (pan) (40D4) (#2920), phospho-Akt (Ser473) (D9E) XP (#4060), PI3 kinase p110α (C73F8) (#4249), cyclin A2 (#91500), and cyclin B1 ( #4135), all purchased from Cell Signaling Technology . Antibodies to CDK4 (#11026-1-AP), CDK6 (#14052-1-AP), SKP2 (#15010-1-AP), p53 (1C12) (#2524), P16-INK4A (#10883-1-AP), EGFR (#18986-1-AP), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (#60004-1-Ig) were purchased from Proteintech . The anti-FLAG was obtained from Sigma-Aldrich . All secondary antibodies were purchased from Cell Signaling Technology, Danvers, MA, USA.Cells were seeded into 6-well plates  at a density of 500 cells/well. After 24 h, cells were treated with or without nobiletin for 48 h. The nobiletin-containing medium was then removed and replaced by complete medium, followed by incubation at 37 °C for 10–14 days. The colonies were fixed with 4% paraformaldehyde, stained with Crystal Violet, and counted. The colonies composed of ≥ 50 cells were counted using a microscope. Each experiment was conducted in triplicate.For transient transfection, the cells were seeded in an antibiotic-free medium at 37 °C for 24 h and transfected with constitutively active SKP2 plasmid or empty vector DNA using Lipofectamine-2000 transfection reagent  according to the manufacturer’s instructions, and treated 48 h after transfection. The lentivirus used for SKP2 silencing was purchased from Genepharma . The cells were transfected with lentivirus according to the manufacturer’s protocol.An annexin V assay was performed according to the manufacturer’s instructions . Nobiletin- and/or palbociclib-treated cells were collected, and used for annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) staining. The samples were then analyzed using flow cytometry within 1 h. Each experiment was conducted in triplicate.Cells were collected at the indicated time points after nobiletin and/or palbociclib treatment and fixed with ice-cold 70% ethanol, then stored at 4 °C overnight. After washing twice with phosphate-buffered saline, FxCycle PI/RNAse staining solution  was used for detection of the cell cycle according to the manufacturer’s protocol. The stained cells were subjected to cell cycle analysis using a flow cytometer and analyzed for cell cycle phases with the C6 Accuri software system . Each experiment was conducted in triplicate.3 cells/well, and the cells were seeded into 96-well plates, followed by 24 h of culture at 37 °C in an atmosphere with 5% CO2, then were treated with various concentrations of palbociclib or nobiletin and maintained in culture for 48 h. After removing the culture medium, the CCK-8 reaction solution was added according to the manufacturer’s instructions, and the absorbance was measured at 450 nm. The half-maximal inhibitory concentration (IC50) value is a critical index of the dose-response curve. Prism statistical software  was used to calculate the IC50 values and to plot dose-response curves. According to IC50 values, the cells was treated with the given concentration of nobiletin, and cultivated for a further 0, 1, 2, 3, and 4 days. At different times after cell plating, the cells were subjected to the CCK-8 assay, according to the manufacturer’s instructions. Each experiment was conducted in triplicate.The Cell Counting Kit-8 (CCK-8) assay  was used to assess the cell viability. The cell concentration was adjusted to 2 × 1029. Briefly, 786-O and 769-P cell lines (5 × 105 cells) were exposed to 100 μM and 25 μM nobiletin for 24 h, respectively, and followed by 24 h treatment with cycloheximide (50 μg/mL)  to stop de novo protein synthesis. SKP2 levels at 0, 4, 8, 12, and 24 h following cycloheximide co-treatment were then determined using IB analysis. Each experiment was conducted in triplicate.Cycloheximide chase analysis was performed as described previously to define the effect of nobiletin on the stability of the SKP2 proteinGAPDH, was used as a loading control. The following forward and reverse primers were used: SKP2 forward-CAGGCCTAAGCTAAATCGAGAG, SKP2 reverse-CTGGCAATGGT GGTGAAATG; GAPDH forward-AG CCTTCTCCATGGTTGGTGAAGAC, GAPDH reverse-CGGAGTCAACGGATTTG GTCGTAT. Each experiment was conducted in triplicate.Total RNA was isolated from control and nobiletin-treated cells using RNAisoPlus , according to the manufacturer’s instructions. Reverse transcription of the extracted RNA to corresponding complementary DNA was performed using a PrimeScript RT reagent Kit with gDNA Eraser (Takara). The qRT-PCR was performed with a QuantiNova™ SYBR® Green PCR Kit  on an Applied Biosystems 7900HT Real-Time PCR System . The housekeeping gene, 30. Briefly, after deparaffinization, rehydration, antigen retrieval, and blocking, the tissue slides were incubated overnight at 4 °C with the indicated antibodies. The following primary antibodies were used: anti-Ki-67 , anti-p27, and anti-cleaved-capase-3.IHC staining of mice tumors was performed as described previously6 786-O cells were mixed 1:1 with Matrigel (BD Biosciences) in a total volume of 200 μL, and were injected subcutaneously into the right flank side of nude mice. Nude mice were treated with vehicle, nobiletin , palbociclib , and nobiletin + palbociclib when the tumor size reached about 100 mm3. Nude mice were euthanized after 21 days of treatment. The growth of tumor was measured twice a week and the average tumor volume (TV) was calculated according to the equation: TV = (L × W2)/2. The protocol was approved by the Laboratory Animal Welfare and Ethics Committee of the Third Military Medical University of China .Four- to six-week-old BALB/c athymic nude mice  were used, with each experimental group consisting of 5 mice. All animal experiments were conducted according to a protocol approved by the university committee for use and care of animals. A total of 2 × 10t-test. P < 0.05 was considered to be statistically significant. Both CalcuSyn software31 and Jin’s formula32 were used to evaluate the synergistic effects of drug combinations. Jin’s formula is Q = Ea + b / (Ea + Eb - Ea × Eb), where Ea + b represented the cell proliferation inhibition rate of the combined drugs, while Ea and Eb represented the rates for each drug, respectively. A value of Q = 0.85–1.15 indicated a simple additive effect, while Q > 1.15 indicated synergism. Combination index (CI) plots were generated using CalcuSyn software .All data are shown as the average ± standard deviation (mean ± SD) and each experiment was independently repeated at least 3 times. All statistical analyses were performed using the GraphPad Prism, version 5.0. The statistical significance of differences between groups was examined by one-way analysis of variance followed by Tukey’s multiple comparison procedure or Student’s Figure 1A. We first performed a CCK-8 assay to identify the effects of nobiletin in RCC and found that nobiletin significantly inhibited cell growth of RCC cell lines in a dose-dependent manner . The results showed that the IC50 response of nobiletin was lower in the 769-P cell line (IC50 = 20.22 μM) and higher in the 786-O cell line (IC50 = 90.48 μM) . We also found that nobiletin inhibited cell proliferation of 786-O and 769-P cell lines in a time-dependent manner .The chemical structure of nobiletin is shown in ) and promoted cell apoptosis in a dose-dependent manner in 786-O and 769-P cell lines . In addition, nobiletin significantly inhibited the colony formation ability of 786-O and 769-P cell lines . Nobiletin may have inhibited cell proliferation by inducing cell cycle arrest and apoptosis.We next determined whether nobiletin affected cell cycle distribution or apoptosis in RCC cells. The flow cytometry assays showed that nobiletin induced accumulation of G1-phase cells . Nobiletin had no effect on CDK2, CDK4, and cyclin D1 levels, but reduced the expressions of p-CDK2, RB, and p-RB levels with increasing doses, in RCC cell lines , indicating that G1/S transition was blocked. Nobiletin also attenuated the expression of cyclin E and p-RB . Our findings suggested that nobiletin induced cell cycle arrest during the G1 phase by increasing P21/p27, but inhibited CDK2 and RB activities. In addition, the decrease in Bcl-2 levels and the increase in Bax levels  confirmed the apoptotic effect of nobiletin on RCC cell lines.To clarify the mechanism of nobiletin involved in cell cycle arrest and apoptosis, we characterized the expression of G1 phase and apoptosis-related proteins upon nobiletin treatment. Consistent with G1-phase arrest, G1-phase checkpoint proteins p21 and p27 were increased by nobiletin treatment  and time-dependent manner . Next, we determined whether nobiletin downregulated SKP2 in a transcriptional and/or post-translational manner. To assess the effects of post-translational regulation, 786-O and 769-P cell lines were treated with nobiletin without or with co-treatment with a protease inhibitor MG-132, followed by SKP2 IB. In both RCC cell lines, MG-132 co-treatment did not rescue SKP2 expression to levels comparable to drug-free controls . To further confirm the SKP2 degradation caused by nobiletin, we conducted cycloheximide chase analyses, showing that cycloheximide only slightly changed the rate of degradation of SKP2 protein in nobiletin-treated RCC cell lines . However, we found that SKP2 mRNA levels were significantly reduced by nobiletin treatment of 786-O and 769-P cell lines . These results confirmed that nobiletin-induced SKP2 protein downregulation was mainly achieved by decreasing its transcriptional level. We also found that the transcription factor, FOXO3A, which is a negative regulator of the SKP2 and SKP2 SCF complex36, was gradually upregulated by escalating doses of nobiletin in 786-O and 769-P cell lines . Based on these results, we speculated that the downregulation of SKP2 may be associated with increasing FOXO3A levels.SKP2, a well-characterized F-box protein, acts as a classic oncogene by promoting proliferation and survival of cancer cells, mainly through targeted degradation of numerous tumor suppressive proteins, including p2128. Our results showed that the dose of palbociclib required to suppress 50% (IC50) of cell proliferation at 48 h was 0.4662 μM, 0.5548 μM, 1.256 μM, and 7.718 μM for the Caki-1, OSRC-2, 769-P, and 786-O cell lines, respectively . To determine the reasons for the higher concentration of IC50 reactions in 786-O and 769-P cell lines, we detected the expressions of related proteins in 5 renal cell lines, namely the Caki-1, 786-O, 769-P, OSRC-2 and HK-2 cell lines. Higher levels of CDK4, CDK6, CDK2, p-CDK2, and SKP2, and lower levels of p27 were found in 786-O and 769-P cell lines . The p27, as a tumor suppressor protein, can affect CDK2 activity28 and is directly degraded by SKP235. The inhibition of RB phosphorylation caused by the CDK4/6 inhibitor, palbociclib, can be reversed by CDK2, which results in drug resistance. We therefore speculated that RCC cells with higher levels of SKP2 might require higher concentrations of palbociclib to achieve a IC50 response. We first overexpressed SKP2 in the Caki-1 and OSRC-2 cell lines, and then treated them with palbociclib for 48 h. The results showed that SKP2 overexpressed Caki-1 and OSRC-2 required higher concentrations of palbociclib to achieve an IC50 response, when compared with the control group (6C). The 786-O cells, a palbociclib highly-responding RCC cell line, was transfected with either control siRNA, or siRNA targeting SKP2 , followed by treatment with palbociclib for 48 h. The dose of palbociclib required to suppress 50% (IC50) of cell proliferation was 7.718 μM for the control group, which was at least 9-fold more than the SKP2 silencing group  (6F). From the IB results, we found that SKP2 overexpression significantly increased p-CDK2 levels by decreasing p27 levels in the Caki-1 and OSRC2 cell lines , and vice versa . Thus, we confirmed that the insensitivity of CDK4/6 inhibitor, palbociclib, was associated with higher SKP2 levels in RCC cells.Palbociclib, a CDK4/6-specific inhibitor, has shown clinical efficacy, but primary or secondary resistance has emerged as a problem37. As we previously discussed, nobiletin inhibited tumor progression by regulating the SKP2-p21/p27-CDK2 axis, and the insensitivity of the CDK4/6 inhibitor, palbociclib, was associated with higher SKP2 levels in RCC cells. Thus, we speculated that a combination of nobiletin and palbociclib may have the effects of dual inhibition of RCC cells. We selected 786-O and 769-P cell lines for the following experiments. Both CalcuSyn software and Jin’s formula were used as previously described to determine the synergy of the 2 agents. The 786-O was cultured with combinations of the 2 drugs at different doses but at a constant ratio  for 48 h. The combination of 6.25 μM nobiletin with 0.625 μM palbociclib in the 786-O cell line inhibited cell proliferation by 32.0%, compared with monotherapy of nobiletin by 15.1% or palbociclib by 11.2%, indicating synergism . Escalating doses, i.e., co-treatment with 12.5 μM nobiletin and 1.25 μM palbociclib  or 25 μM nobiletin and 2.5 μM palbociclib  or 50 μM nobiletin and 5.0 μM palbociclib , showed synergetic effects in 786-O cells . Those findings showed that nobiletin and palbociclib had synergistic lethality, because they inhibited both CDK2 and CDK4/6 kinases. Furthermore, a combination of the 2 agents strongly induced apoptosis, when compared with a single agent in the 786-O and 769-P cell lines , which was further demonstrated by the increase of cleavage of caspase-8 and caspase-3 . Similarly, a significant increase of p27 was observed with the combination treatment when compared with single agent , suggesting that simultaneously inhibiting CDK2 and CDK4/6 kinases did more to induce apoptosis.Clinical trials are currently ongoing with the single agent palbociclib in several advanced solid tumorsin vitro findings by using an in vivo xenograft model. A 786-O xenograft model was established and treated with vehicle, nobiletin, and/or palbociclib. Consistent with the in vitro results, the combination of nobiletin and palbociclib suppressed tumor growth significantly more than single agent treatment . The average tumor size and tumor weight at the end of the experiment (treatment for 21 days) were significantly lower in the nobiletin-palbociclib combination group (8C). The body weight of the xenograft model was unchanged during drug treatment, suggesting that the effect on normal tissues was minimal . Finally, immunohistochemical staining of tumor tissues revealed that compared with nobiletin or palbociclib single agent treatment, a combination of the two agents inhibited cell growth (decrease of Ki-67 and increase of p27) and induced apoptosis (increase of cleaved caspase-3)  significantly more.We validated the above 38, making them potential antitumor drugs7. Nobiletin is a common PMF that has been reported to prevent various tumor inductions and progressions40. Several studies have reported the antitumor activities of nobiletin, including inhibition of proliferation, induction of the cell cycle arrest, and promotion of apoptosis42.Polymethoxylated flavonoids (PMFs) have a variety of biological activities such as anti-cancer, anti-cardiovascular disease, prevention and treatment of obesity, and anti-inflammatory activitiesFigure 1) by inducing G1-S phase arrest in RCC cells . The causal role of nobiletin in G1/S arrest was due to an increase of p27 and p21 and inhibition of CDK2 phosphatase, ultimately leading to a decrease of p-RB . The p21 and p27 are crucial for restraining the G1-S phase transition43, and are regulated by the E3 ubiquitin ligase, SKP235. Nobiletin caused accumulation of p27 and p21 levels by downregulating SKP2 protein expression in time- and dose-dependent manners by decreasing its transcriptional level , which might be due to nobiletin-induced upregulation of FOXO3A. FOXO3A is a transcriptional repressor of SKP2 gene expression, which directly binds to the SKP2 promoter36. Small molecules targeting SKP2 activity or SKP2 complex assembly have been examined in leukemia cells and in xenograft tumor models, respectively, and have been proven to be effective as anticancer agents45. These findings not only provide new insights into the molecular understanding of nobiletin’s anti-proliferative effects, but also highlight the potential of nobiletin in the development of SKP2-targeted anticancer therapeutics.In the present study, we found that nobiletin inhibited tumor proliferation and progression in RCC by regulating the SKP2-p21/p27-CDK2 axis. Nobiletin inhibited the proliferation of RCC lines in dose- and time-dependent manners  and OSRC-2 (IC50 = 0.5548 μM), but showed less effects in 769-P (IC50 = 1.256 μM) and 786-O (IC50 = 7.718 μM) cells, especially the 786-O cell line . The insensitivities to 786-O and 769-P may be explained by the higher levels of p-CDK2 and SKP2 but lower levels of p27 . CDK2/cyclinE could be deactivated by p2728, which could be targeted for degradation by SKP235. A previous study confirmed that while estrogen receptor- (ER) positive breast cancer cell lines were inhibited by palbociclib, they quickly adapted because of degradation of p27 and a subsequent activation of CDK2, allowing compensatory phosphorylation of RB and passage into the S phase28. Another study also confirmed that insensitivity to CDK4/6 blockade was mediated by p-RB phosphorylation recovery induced by the noncanonical CDK2/cyclinD1 complex27. The cyclinE rebound is likely a consequence of CDK2/cyclinD1 activity and eventually triggering S-phase entry27. Based on these observations, searching for agents that reverse resistance of palbociclib represents a promising strategy to discover novel cancer therapeutics46. Herein, we showed that nobiletin downregulated SKP2 levels to suppress activation of CDK2 phosphatase . A combination of nobiletin with palbociclib further inhibited cell proliferation and induced cell apoptosis by increasing p27 and proapoptotic protein levels, when compared to palbociclib treatment alone . Similarly, a combination of nobiletin and palbociclib significantly inhibited tumor growth in a xenograft model (8E).49. It is also noteworthy that some natural compounds have been widely used in clinics, such as paclitaxel50. Nobiletin, as a natural extract, can be considered as a promising candidate for cancer therapy due to its multiple-targeting capabilities51. But the underlying mechanisms and bioavailability of nobiletin are still complex problems to understand52, which limits its application as a therapeutic agent. Our study demonstrated that nobiletin inhibited tumor progression by regulating the SKP2-p21/p27-CDK2-RB axis : SKP2 targets p27 for degradation, CDK2 can be reactivated upon p27 degradation, and phosphorylation of RB is recovered by activated CDK2, which leads to G1/S transition, tumor growth, and progression. However, nobiletin decreased SKP2 protein levels by reducing its transcriptional levels, and showed synergistic chemopreventive effects with palbociclib in RCC cells. The fact that a combination of nobiletin and palbociclib at their half doses produced stronger anti-cancer effects than nobiletin or palbociclib alone, provided the strong basis to utilize the nobiletin/palbociclib combination for renal cell carcinoma chemoprevention.It is noteworthy that natural compounds improved the sensitivity and efficacy of chemotherapy drugsNobiletin downregulated the SKP2-p21/p27-CDK2 axis to inhibit tumor growth and progression in RCC cells. A combination of nobiletin with a CDK4/6 inhibitor may be a new therapeutic strategy to overcome the resistance of a single agent CDK4/6 inhibitor in cancer  treatments.Click here for additional data file.