Psidium guajava leaves present a diverse phytochemical composition including flavonoids, phenolics, meroterpenoids, and triterpenes as the major bioactive constituents. Guajadial, a caryophyllene-based meroterpenoid, has been studied for potential anticancer effects tested in tumor cells and animal experimental models. Moreover, guajadial has been reported to have a mechanism of action similar to tamoxifen, suggesting this compound as a promisor phytoestrogen-based therapeutic agent. Herein, the anti-estrogenic action and anti-proliferative activity of guajadial is reported. The enriched guajadial fraction was obtained by sequential chromatographic techniques from the crude P. guajava dichloromethane extract showing promising anti-proliferative activity in vitro with selectivity for human breast cancer cell lines MCF-7 and MCF-7 BUS . Furthermore, evaluation of anti-estrogenic activity in vivo was performed demonstrating that guajadial enriched fraction inhibited the proliferative effect of estradiol on the uterus of pre-pubescent rats. These results suggest a relationship between anti-proliferative and anti-estrogenic activity of guajadial, which possibly acts in tumor inhibition through estrogen receptors due to the compounds structural similarity to tamoxifen.The research of natural products has allowed for the discovery of biologically relevant compounds inspired by plant secondary metabolites, which contributes to the development of many chemotherapeutic drugs used in cancer treatment.  Catharanthus roseus (Vinca rosea) demonstrating to have potent cytotoxic effects [Cancer is one of the leading causes of death worldwide , with an effects .Psidium guajava L. (Myrtaceae), a native species from tropical areas, has been reported as a good source of secondary metabolites with pharmacological activity, such as flavonoids, terpenoids, phenols, tannins [P. guajava extracts and oils are reported to display anti-inflammatory [ tannins ,6. Amongammatory , antibacammatory , antidiaammatory , anticouammatory . The cytammatory ,13,14,151), psidial A (2), psiguadial A and B presented significant in vitro anti-proliferative effect against nine human cancer lines [P. guajava leaves extract [1 and 2 may act as selective estrogen receptors modulators (SERMs) [Psidium guajava L. leaves extract suggesting a possible relationship between anti-proliferative activity and hormonal action. This correlation was confirmed both by E-screen tests and in vivo tests that assessed uterotrophic activity.Our research group have demonstrated that guava’s dichloromethane leaves extract and the mixture of guajadial  . NeverthHexane, dichloromethane, methanol, acetic acid, p-anisaldehyde, sulfuric acid, dimethyl sulfoxide, trichloroacetic acid, doxorubicin, and colchicine were commercially purchased from Sigma Aldrich.Column chromatography (CC): silica gel 60 , 2 × 50 cm). TLC (thin-layer chromatography): precoated plates , UV detection, and anisaldehyde solution.Psidium guajava L. were collected at CPQBA–Unicamp  experimental field. The dried specimen was deposited at Herbarium number 1335 under authorization of Brazilian Genetic Resources (SisGen) (010003/2013-3).P. guajava leaves were ground with dry ice and extracted with dichloromethane  by mechanical stirring  at room temperature. The partial extracts were combined and the organic solvent was eliminated under vacuum by rotary evaporation system at 40 °C . The resulting dichloromethanic crude extract (DCE) was weighted and kept under refrigeration. Then, DCE (35 g) was homogenized with diatomaceous earth Celite® 545   and the mixture was fractionated by adsorption liquid chromatography over Silica gel 60  0.0063–0.200 mm thickness. Elutent polarity was increased by gradients of hexane, dichloromethane, and methanol. The 50 mL fractions were collected. Obtained fractions were grouped according to thin-layer chromatography (TLC) profile ) followed by heating at 110 °C, resulting in three fractions named A, B, and C , and methanol. The 2 mL fractions were collected, resulting in five fractions  after grouping by TLC profile (B3 fraction (1.6 g) was fractionated on reverse phase Phenomenex  column C-18  with eluting mobile phase of deionized water, acetonitrile/water mixture  and was named FFINAL since was chemically characterized by GC-MS and contained the active compounds 1 and 2. Thus, FFINAL was tested by in vitro and in vivo assays.Fractions B and B3 were selected for further fractionation after gas chromatography–mass spectrometry (GC-MS) analysis indicating the presence of compound −1)–240 °C (20 min), injector 220 °C, detector 250 °C. Helium was used as a carrier gas . The MS were taken at 70 eV. Sample volume was 1–2 μL. The mass spectra were compared with software NIST05 MS Search.The GC/MS analysis was carried out using Agilent  gas chromatograph HP-6890/5975 system equipped with an HP-5 (30 m × 0.25 mm × 0.25 μm). Temperature program: 60 °C (3 °C·minv/v) penicillin: streptomycin  in a humidified atmosphere with 5% CO2, at 37 °C. For the experiments, cell lines were used between passages 4 to 12.Human tumor and immortalized cell lines  were grov/v) penicillin:streptomycin ). Diluted as described for samples, Doxorubicin  was used as positive control on anti-proliferative assay while Colchicine  was the positive control on cell cycle assay.The DCE and resulting fractions (5 mg) were initially diluted in dimethyl sulfoxide (DMSO) (50 µL) followed by the addition of complete medium (950 µL). Final concentrations  were obtained by serial dilution in complete medium, except for E-screen assay. For E-screen experiment, it was used a low estrogen medium  supplemented with 5% Dextran-charcoal-treated fetal bovine serum , and 1%  did not affect cell viability. Before (T0) and after (T1) treatment, cells were fixed with trichloroacetic acid  and cell proliferation determined by spectrophotometric quantification at 540 nm using sulforhodamine B dye [® (OriginLab Corporation) [Cells in 96-well plates  (100 µL wellne B dye . Using toration) .−1), weak activity (15 μg·mL−1 < TGI < 50 μg·mL−1), moderate activity (6.25 μg·mL−1 < TGI < 15 μg·mL−1), and potent activity (TGI < 6.25 μg·mL−1)) was adopted to select the most active fraction to be evaluated in further in vitro and in vivo experiments.For comparative evaluation among resulting fractions of DE, the criteria described by Fouche et al. (2008)  (inactiv4 cell mL−1) in 12-well plates were incubated with low estrogen medium (2 mL well−1) for 72 h. After that, cells were treated with DCE (5 and 25 μg·mL−1) or FFINAL fraction  in absence or presence of 17-β-Estradiol  and incubated for 144 h. Cell viability were determined by spectrophotometric quantification (540 nm) of cellular protein content using sulforhodamine B (SRB) assay, as described for anti-proliferative assay.MCF7 BUS cells  were seeded on 12-well microplates and, after 24 h, were submitted to cell cycle synchronization by FBS suppression. After more 24 h, cells were treated with FFINAL fraction (2.5 and 5 µg·mL−1) and incubated at 37 °C, 5% CO2 for 24 and 48 h. Colchicine (0.25 μM) was used as positive control. Afterward, cells were trypsinized (trypsin-EDTA 0.25%), washed with Phosphate-buffered saline (PBS) and resuspended in 500 µL of cold ethanol 70%. After 12 h at 4 °C, cells were washed with PBS and resuspended in 200 µL of Guava cell cycle 4500-0220  for 30 min at room temperature and in the dark. Data acquisition (5.000 events per replicate) and analysis were performed using FACS- Canto II cytometer , at the Central Laboratory of High Performance Technologies (LaCTAD)/UNICAMP.MCF7 BUS cells  and Balb-C female mice , aged 8–10 weeks and weighing 20–35 g, and Wistar female rats, aged 21 days, were used for in vivo assays. The animals were obtained from the Multidisciplinary Center for Biological Investigation on Laboratory Animal Science (CEMIB/UNICAMP) after experimental protocols been approved by the institutional Committee for Ethics in Animal Research, at the University of Campinas . All procedures were in accordance with the principles and guidelines adopted by Brazilian College of Animal Experimentation (COBEA).®, Curitiba, Brazil) and potable water.Animals were housed (5 mice or rats per cage) in polyethylene boxes (matte white) with disinfected softwood beddings, at room temperature of 22 ± 2 °C; relative humidity of 50 ± 20% and light/dark cycle of 12 h/12 h, with free access to food   and dissolved in PBS (pH 7.0). Vehicle was PBS, pH 7.0 + Tween 80®  0.3%.FFINAL fraction  or vehicle . All animals were observed during 4 h and then daily for 14 days, for general toxicity signals evaluation: bodyweight evolution, locomotion, behaviour , respiration, salivation, tearing eyes, cyanosis, and mortality. At the 14th experimental day, animals were euthanized by deepening anesthesia with a mixture of ketamine  and xylazine  followed by cervical dislocation [Fifteen Swiss female mice were fasted for 4 h and then treated orally with F5 tumor cells/animal in PBS . For testing, cells were prepared at a density of 5 × 106 cells/50 µL/animal in PBS after cell viability evaluation with trypan blue in Neubauer chamber.Ehrlich tumor cells were maintained in the ascitic form, until 20 passages, by peritoneal passages in Swiss male mice by weekly transplantation of 5 × 106 cells/50 µL/animal) was inoculated subcutaneously in the flank of Balb/C female mice . On the 3rd day, animals (40 mice) were randomly distributed into negative ), positive ) and experimental  groups. One group, named Satellite , were not inoculated with Ehrlich cells suspension neither treated with any sample. Animals on vehicle and experimental groups were treated orally alternately and those of doxorubicin group were treated intraperitonially every three days during the 12 days. Clinical parameters  were evaluated every treatment day and bodyweight was recorded at days 0 and 16th experimental day. On 16th day after anesthesia, blood samples  were collected from each animal by the retrorbital plexus for blood counting . Then, animals were euthanized by deepening anesthesia with a mixture of ketamine  and xylazine  followed by cervical dislocation. Necropsies were performed on each mouse to assess gross toxicity to some organs  and to remove the tumor mass. The relative tumor weight (RTW) was calculated as tumor weight divided by final corporal weight. The growth inhibition ratio was calculated as /mean RTWnegative control) × 100 [Ehrlich cells suspension (5 × 10l) × 100 .v/v) penicillin:streptomycin (1000 U/mL:1000 mg/mL)), at 1 × 105 cell/160 μL density. The cells were then gently infused into sterile conditioned polyvinylidene fluoride hollow fibers (Spectrum Laboratories) , the fibers were heat-sealed and cut at 2.0 cm intervals. Prior implantation, the fibers were incubated for 24 h in working medium in a humidified atmosphere with 5% CO2, at 37 °C. After that, the fibers were randomly separated into groups. Six fibers fragments were transferred to 6-well plate (1 fragment per well) containing Thiazolyl Blue Tetrazolium Bromide (MTT) solution . After 4 h-incubation, the absorbance (550 nm) was measured after formazan dilution with DMSO (250 µL/well) to establish the basal value (D0).Confluent MCF7 cells were harvested with tripsin/EDTA and suspended in working medium ,  and 1%  into the peritoneal cavity of immunocompetent Balb-C female mice  under anesthesia . At 3rd day, animals (25 mice) were randomly distributed into five experimental groups that were treated during seven days with vehicle , daily), doxorubicin  and FFINAL fraction . At 8th day (D8), animals were euthanized by deepening anesthesia with a mixture of ketamine  and xylazine  followed by cervical dislocation. Necropsies were performed on each mouse to assess gross toxicity to major organs. Then the fibers were retrieved, placed into Petri dishes containing working medium and kept at 37 °C for 30 min. After that, fibers were transferred for a Petri dishes containing MTT (1.25 mg/mL) in working medium, incubated for 4 h in a humidified atmosphere with 5% CO2, at 37 °C, and transferred to Petri dishes containing protamine sulfate solution 2.5% . To spectrophotometric evaluation, fibers were cleaned, air-dried (24 h) and cut in half before formazan extraction  with DMSO (250 µL/fiber). Aliquots (100 µL/well) were transferred into 96-well plate and optical density (O.D.) were measured at 550 nm. Results were expressed for each group as ((average O.D.D8 − average O.D.D0)/average O.D.D0) × 100 [The second group of fiber fragments were transplanted (0) × 100 ,23,24.n = 8 animals/group). Positive and negative control groups were orally treated with 17β-estradiol (0.3 mg/kg) and vehicle (5 mL/kg), respectively. The other three groups were treated with 17β-estradiol  followed by FFINAL fraction  treatment for 3 days. Bodyweight was recorded at days 0 and 4th experimental days. On the 4th day, animals were euthanized by deepening anesthesia followed by cervical dislocation and uteri and ovaries were rapidly collected, stripped of adipose tissue and weighted for calculating uterine and ovary relative weight [Forty Wistar female rats 21–25 days) were randomly distributed into 5 experimental groups  followed by Tukey’s test or Two-way ANOVA followed by Bonferroni test, depending on the most appropriate test in each case. The tests are described in the results. Statistical analyses were performed with the GraphPad Prism 5 software .FINAL showed the presence of three major compounds (retention time (RT): 28, 45 and 46 min)  . The nor1 and 2, isomeric natural products isolated from guava leaves [m/z 474, 446, 405, 391, 309, 271, and 241  and prostate  tumor cell lines. After successive fractionation, fraction FFINAL showed potent activity  against ovary , lung , prostate , glioma , breast  and multi-resistant ovary  tumor cell lines (DCE showed weak anti-proliferative activity against ovary (TGI = 27.23 μg·mLll lines ).FINAL was selected for further in vitro anti-proliferative assay against a panel of human breast tumor cells and non-tumor cells (MCF-7 (estrogen-dependent mammary adenocarcinoma), MCF-7 BUS (mammary adenocarcinoma overexpressing estrogen receptor), MDA-MB 231 (negative triple mammary adenocarcinoma) and MCF-710A ). FFINAL showed potent anti-proliferative activity against MCF-7 BUS (TGI = 2.27 µg·mL−1) and MDA MB 231 (TGI = 5.13 µg·mL−1) cells  cells ). The cho et al. , which dBreast cell lines: MCF-7 (express the estrogen and progesterone receptor); MCF-7 BUS (estradiol receptor overexpressing); MDA MB 231 ; MCF 10A (non-carcinogenic cell line). TGI: Total Growth Inhibition.FINAL, evaluating the estrogenic activity based on the effects of FFINAL in the presence and absence of 17β-estradiol . FFINAL concentrations were determined based on TGI values for MCF7 BUS cell line (p < 0.001) after treatment with 17β-estradiol (positive control) comparing to the negative control (cells and medium), indicating the test was valid.This quantitative test compares cell growth of 17β-estradiol-treated MCF-7 BUS cell line and cultures treated with different concentrations of the Fell line . MCF7 BU−1 DCE totally inhibited estrogen action, while DCE-treated cells at 25 µg·mL−1 presented a limited increasing proliferation when stimulated by E2. This inversely proportional effect may indicate that DCE act as a partial estrogen receptor agonist.DCE inhibited cell proliferation of E2 with inverse relationship to concentration , i.e., aFINAL fraction at different concentrations  were evaluated  of FFINAL significantly inhibited the proliferative effect of E2, indicating a possible anti-estrogenic activity.Fvaluated . In the Flow cytometry evaluates cells proportion in a population on several cell cycle stages representing a valuable tool to study mechanisms of action of substances with anticancer potential .FINAL fraction on the MCF-7 BUS cell cycle was evaluated with two concentrations (2.5 and 5.0 µg·mL−1) during 24 and 48 h of treatment (duplicate in each treatment). Colchicine (0.25 μg·mL−1) was employed as positive control and caused an increase in G1 MCF-7 BUS cell population after 24 h of treatment (FINAL fraction (5 µg·mL−1) varied MCF-7 BUS cell population after 24 h of treatment, increasing significantly (p < 0.001) G1 phase of treated cell (G1—71.54%) compared to cell control (G1—18.06%). Furthermore, a significant reduction (p < 0.001) of the populations in both S phase (19.7%) and G2/M phase (8.6%) (p < 0.05) related to the cell control group (61.2%—S and 20.69%—G2/M) was observed. The treatment of MCF-7 BUS cells with FFINAL fraction (2.5 µg·mL−1) significantly interfered (p < 0.01) in G1 and G2/M phases, with a similar profile to cell control only in S phase of the cycle.Therefore, the activity of Freatment A. FFINALFINAL fraction concentrations at 2.5 and 5.0 µg·mL−1 significantly increased (p < 0.001) G1 phase cell population  after 48 h, when compared to the cell control group (46.9%) (F (46.9%) B. ColchiFINAL fraction concentrations of 2.5 µg·mL−1 and 5.0 µg·mL−1 .To evaluate whether in vivo experimental models would reproduce the in vitro anti-proliferative activity observed for F−1 of FFINAL fraction-induced piloerection, abdominal writhing and lethargy. The most intense signs of toxicity were observed in animals treated with the highest dose than those receiving the lowest dose. All animals recovered after 24 h, with no deaths. Furthermore, no significant difference on the relative organs weight (control and treatment groups) were observed. Therefore, 50 mg·kg−1 dose of FFINAL fraction were considered safe as the maximum dose in Ehrlich solid tumor and Hollow Fiber models.During the first 4 h after treatment, both 200 and 100 mg·kgFINAL was evaluated in flank tumor models induced by injection of Ehrlich murine tumor cells. The purpose of this test was to evaluate whether oral samples would reduce the development of solid Ehrlich tumor [In vivo studies were performed with Ehrlich solid tumor on Swiss mice. The anticancer activity of Fch tumor .FINAL fraction-treated groups showed mean tumor weight of 0.045 ± 0.007 g, 0.041 ± 0.009 g and 0.058 ± 0.013 g at 12.5, 25 and 50 mg·kg−1, respectively. The average tumor weight of doxorubicin control group was 0.027 ± 0.01 g. Considering the relative tumor weight , FFINAL fraction oral treatment significantly (p < 0.001) inhibited tumor growth, dose-independently, as well as doxorubicin (p < 0.001) comparing to the vehicle group  of experiment, presented clinical signs of toxicity. These signs included piloerection, secretion of tears and pain, along bodyweight loss (FINAL-treated groups (50 and 25 mg·kg−1). Therefore, the animals of these groups  did not receive treatment until last day of the experiment (19th day). The interruption in induced bodyweight loss suggesting reversibility of toxicity signs. More, none animal treated with 12.5 mg·kg−1 of FFINAL fraction showed any toxicity signs.During the experiment, the animals treated with 25 and 50 mg·kgght loss , resultiFINAL fraction, despite clinical signs of toxicity of this group.The total erythrocyte counts as well as hemoglobin, hematocrit and mean corpuscular volume values were altered in the doxorubicin-treated animals among hematological parameters studied at the end of the experiment . These r−1) of FFINAL had a significant increase in relative weight (p < 0.001). Furthermore, the relative uterus weight of doxorubicin-treated animals decreased in comparison to the vehicle group (p < 0.05) while animals treated with the highest doses of FFINAL showed an increase in the relative ovaries weight in comparison to the vehicle group (p < 0.001).Some organs were weighed and macroscopically observed for possible signs of toxicity, such as weight gain . CompariFINAL fraction in in vitro anti-proliferative test, this cell line was selected to perform Hollow Fiber assay.Hollow fiber is a method used to propagate human cancer cells in inert fibers with small pores; enough to retain cancer cells but large enough to allow permeability of potential chemotherapeutic drugs . As the FINAL fraction  (−1) being the most efficient.By comparing cell amounts inside the fibers at the beginning and at the end of the test, in the vehicle group (negative control) was observed cell proliferation of 13.5%. Doxorubicin treatment presented cytocidal activity of −50.5% as well as all doses of Fctively) . These rFINAL fraction at 50 mg·kg−1 that promoted negative bodyweight variation at the 4th experimental day.As expected by the results of Ehrlich solid tumor assay, doxorubicin treatment induced bodyweight loss at the 7th experimental day, as well as the FFINAL fraction reduced animal weights at the highest dose at the beginning of treatments indicating animal bodyweight variation .F−1, orally) for three days significantly increased (p < 0.001) relative uterus weight of prepubertal rats when compared to the vehicle group, which is in agreement with the literature [FINAL (after 15 min of E2 administration), was able to significantly inhibit the proliferative effect of E2, regardless of FFINAL concentration  was able to suppress the proliferative effect of 17β-estradiol on the MCF7 BUS cell line.As expected, the treatment with 17β-estradiol , suggesting a possible relationship between anti-proliferative activity and hormonal action. This correlation was confirmed both with E-screen and in vivo tests in uterus of prepubertal rats.This work investigated the anticancer and anti-estrogenic action of extract and fractions obtained from FINAL with a well-established estrogenic profile being sensitive to estradiol and therefore, most suitable for evaluating anti-estrogenic activity. Rizzo et al. [P. guajava leaves.The choice of MCF7 BUS cell line to continue mechanism of action studies, considered the high sensitivity towards treatment with Fo et al.  reportedP. guajava extract leaves increased estradiol and progesterone levels in female rats, by 17.6 and 34.6% respectively. These observations indicated a great hormonal potential with possible action on estrogen receptors that prompted investigation in order to confirm such activity. Our data demonstrated, FFINAL to significantly inhibit proliferative effect of estradiol on MCF7 BUS breast cells at the two highest concentrations tested (1.25 and 2.5 μg·mL−1), suggesting a straight relationship of effect with sample concentration.Uboh et al.  reportedFINAL fraction presented compounds with a similar action to drugs which compete with estrogens in their receptor connection causing an inhibitory effect on cell proliferation. There is a valuable improvement in the process of drug development when the activity is associated with a specific receptor. The classification of receptors based on pharmacological responses remains a valuable and widely used approach [Tumor cells that express estrogen receptors (ERs) indicate that cell proliferation depends on estrogen stimulation. Therefore, hormonal block may be a possible alternative to reduce cell growth. There are two alternatives to treatments available in this case: reduction of estrogens normally produced by the body and inhibition of connections between receptors and hormones. E-screen test result indicated that Fapproach .FINAL treatment. Therefore, evaluation of FFINAL activity on cell cycle was performed by flow cytometry. FFINAL stimulated a significant increase in G1 cell populations in both concentrations of 2.5 µg·mL−1 and 5.0 µg·mL−1, after 24 and 48 h, indicating possible impediment of cells to proceed to S phase. These results suggested that anticancer effect of guajadial enriched fraction does not involve exclusively the action on estrogenic receptors, but also cell death mechanism activation initiated by exposure of phosphatidylserine residues and interruption G1-phase cell cycle. The tamoxifen mechanism of action is well-established on cancer cells and consists in inhibiting DNA synthesis, inducing apoptosis and blocking cell cycle in G1 phase [FINAL fraction exhibited an activity on G1 phase of MCF-7 BUS cell cycle.One of the most striking features of cancer cells is the loss of cell division control, caused by genetic changes that alter this process. The main strategies of mechanisms used in cancer therapies development are reduction of tumor growth and induction of cell death . The antG1 phase . SimilarFINAL after 15 min of estradiol (E2) administration was able to significantly inhibit proliferative effect on uterus, however, did not alter the relative weight of the prepubertal rats ovaries. This fact can be explained through the hormones involved in this process. Pituitary hormones FSH (follicle-stimulating hormone) and LH (luteinizing hormone) are related to ovarian stimulation and menstrual cycle. Hypothalamus regulates the release of pituitary hormones and is regulated by estrogen and progesterone (produced by the ovaries). Elevated levels of estrogens and progesterone stop the pituitary gland stimulation and therefore, blocking estrous cycle and consequently inhibiting ovulation. In prepubertal animals, this cycle is not yet active and, therefore, the ovary is not producing estrogens, which would influence the test results. The ovaries are mainly influenced by chorionic gonadotropin (produced by placenta), FSH and LH during prepubertal phase. These hormones, when injected into the prepubertal rat, will produce ovarian stimulation characterized by increased blood irrigation. In the test used, there would be changes in the ovaries only if the guava’s active ingredients had an effect on the FSH and LH ovarian receptors [The treatment with Feceptors ,39.P. guajava fraction evidenced by anti-proliferative activity in vitro. In this study, oral treatment with FFINAL  resulted in significant inhibition of solid tumors development and human tumor cells confined in semi-permeable fibers, as well as doxorubicin at a concentration of 3 mg·kg−1, demonstrating efficacy in solid tumors treatment. The effect produced by oral administration of extracts and fractions of P. guajava is highly relevant for the future development of a product, which could be an alternative to the chronic treatment carried out with tamoxifen and estrogenic antagonists.There are limitations related to cell culture such as predicting chemotherapy-response of solid tumors. Therefore, development of experimental models is crucial in order to enhance in vitro drug screening . In thisPsidium guajava L. as potential source of anticancer molecules. The enriched guajadial fraction exhibited promising anti-proliferative activity in vitro with selectivity for human breast cancer cell lines MCF-7 and MCF-7 BUS . Furthermore, for the first time, anti-estrogenic activity in vivo of guajadial was investigated demonstrating that guajadial enriched fraction inhibited proliferative effect of estradiol on the uterus of pre-pubescent rats. These results indicate that guajadial acts in tumor inhibition through estrogen receptors, suggesting a tamoxifen-like mechanism of action. Therefore, Psidium guajava L. could be a promising source for development of new cancer treatments with further studies required to better understand cellular and molecular mechanisms of anti-cancer and anti-estrogenic action.The exploration of active compounds present in medicinal plants has become extremely essential in phytochemical studies associated with pharmacology. Guava leaves have been reported in the literature as promising natural source of anticancer agents. Results reported herein, using an enriched guajadial fraction, support the relevance of


To evaluate titanium (Ti) sputtering of the poly(methyl methacrylate) (PMMA) stem of the Boston Keratoprosthesis (BK) as a method to enhance interfacial adhesion between the PMMA and the recipient corneal tissue.2 and coated with Ti using a DC magnetron sputtering instrument. The topography and hydrophilicity of the surfaces were characterized using atomic force microscopy and a water contact angle instrument, respectively. Scratch hardness and adhesion of the Ti film were measured using a mechanical tester. Biocompatibility assessments were performed using cultured human corneal fibroblasts and whole blood ex vivo. The optical quality of the Ti sputtered BK was evaluated using a custom-made optical bench.PMMA specimens were plasma treated with Ar/OBy contact angle studies, the Ti coating improved PMMA hydrophilicity to match that of medical-grade Ti (Ti-6Al-4V-ELI). Ti sputtering of contact surfaces resulted in a plate-like morphology with increased surface roughness, without impacting the transparency of the BK optical component. Scratch testing indicated that the mechanical behavior of the Ti coating was similar to that of casted Ti, and the coating was stable in pull-off adhesion testing. Sputtered Ti film was highly biocompatible based on tests of cell viability, adhesion, proliferation, differentiation, collagen deposition, and keratocan expression, the properties of which exceeded those of uncoated PMMA and did not induce increased complement activation.Titanium coating of the BK stem generated a mechanically and biologically favorable interface, which may help to enhance corneal stromal adhesion and biocompatibility.Improving the biocompatibility of the BK PMMA stem may improve long-term outcomes of implantation. Plasma treatment was carried out using Femto Plasma Cleaner  at 100 W for 2 minutes under an argon/oxygen 80/20 mixture to produce plasma-treated PMMA, or (p)PMMA. Immediately after surface treatment, PMMA discs or the BK with the stems facing down (anterior down) were taped to the sample holder using Kapton tape, and a thin Ti film was deposited on the PMMA substrates, PMMA and (p)PMMA, using a DC magnetron sputtering instrument  to generate Ti-coated PMMA (Ti-PMMA) and plasma-treated then Ti-coated PMMA (Ti-(p)PMMA), respectively. The sputtering chamber was pumped to a vacuum level below 2.0 × 10–6 Torr prior to coating. During the deposition, the sample holder was rotated at 40 rpm. The distance between the substrate holder and the sputtering target (Ti with a purity of 99.999%) was set at 15.0 cm. Deposition was conducted under 200-W DC power with pressure of 4 × 10–3 Torr with argon gas for 2 hours at a temperature of 40°C. To protect the optical components, the front and back  were covered with 5- and 2.5-mm-diameter tapes, before plasma treatment, to be removed after Ti deposition. The thickness of Ti layer was measured by a profilometer  by assessing the height difference between the deposited layer and the substrate on the PMMA discs from multiple points (n = 8), with the exclusion of the wall-type features of the Ti film. Titanium discs of 15.6-mm diameter were prepared using Ti-6Al-4V-ELI medical-grade Ti (grade 23) of 580-nm thickness, cleaned and polished prior to use.Medical-grade PMMA  discs with 0.5-mm thickness and 40.0-mm diameter were cut to 8.0-mm diameter by a laser cutter .The topography of the surface of the PMMA discs was probed using an Asylum Research Cypher atomic force microscope  in AC mode using a standard AFM probe . Topographical surface data were acquired in phase and height profiles with scan sizes of 30 × 30 and 2 × 2 µmn = 8 per group).Surface hydrophilicity of the Ti coating on the PMMA discs was assessed with a contact angle and surface tension measurement system  using a static sessile drop technique. At room temperature, a 5-µL droplet of distilled water was deposited by a syringe located above the sample surface, and the image was captured from the side by a high-resolution camera. The contact angle for each group was analyzed using FTA 2.1 software and averaged for each group (X-ray photoelectron spectroscopy (XPS) spectra of the PMMA discs (before and after sputtering) were acquired in the range from 100 to 1300 eV using a Thermo Scientific K-Alpha spectrometer . Spectra were obtained via Avantage software (Thermo Fisher Scientific) in the survey and high-resolution modes with an energy step size of 1 eV and 10 scans.n = 7) were individually held in a diaphragm in front of the microscope located 304.8 cm away from an illuminated 1951 USAF resolution test chart .Transparency-resolution studies of the BK before and after Ti coating were carried out using an optical bench, as previously described.n = 8 per group).The imputed visual acuity was extracted after converting the element numbers of the resolution chart into their corresponding optotypes, based on distance and angle of the optotype bars as previously described.2). Surface contaminants were washed off the stud using a detergent solution followed by acetone. The stud was adhered to an 8-mm-diameter sample or PMMA, Ti-PMMA, or Ti-(p)PMMA with a thermally curable epoxy adhesive  and cured at 50°C for 6 hours; in the stability study, the samples were soaked in Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F-12), 1:1 , for a week before the measurement. The tensile strength of the Ti coating was assessed by measuring the mechanical force required to separate the Ti film from the substrate (n = 8 per group).The adhesion strength between the Ti coating and the PMMA discs was tested with the pull-off method using a mechanical tester  according to the ASTM D4541-17 standard.n = 8 per group). In the stability study, the samples were soaked in cell culture media 2 for 1 week before measurement.The scratch resistance of Ti-coated PMMA discs was assessed with increasing normal load2 for up to 7 days. The cell culture media were changed every other day. After 1, 4, and 7 days of cell culture, live/dead staining was performed using an Invitrogen LIVE/DEAD staining kit (Thermo Fisher Scientific) composed of calcein acetoxymethyl and ethidium homodimer-1, and imaging was performed using an inverted fluorescent microscope  with 10× objective. Four samples per group were tested and compared to those of tissue culture well plate (TCP) and medical-grade casted Ti discs as positive controls. Four images obtained from each sample were analyzed by ImageJ software  to assess cell viability and confluency as described elsewhere.–To evaluate biocompatibility, 8.0-mm-diameter Ti-(p)PMMA and PMMA discs were fitted to 48-cell culture well plates in 1.0-mL penicillin/streptomycin 5× solution for 15 minutes and then washed with sterile PBS. Then, 5000 human corneal fibroblasts (HCFs), cultured from deceased human corneal donors, were seeded on each disc in 500 µL of DMEM/F-12 media supplemented with 10% fetal bovine serumThe metabolic activity of HCFs cultured on Ti-(p)PMMA discs was assessed using the Invitrogen alamarBlue assay (Thermo Fisher Scientific). HCF cells were cultured on the discs as above. The alamarBlue assay was performed at days 1, 4, and 7 days of culture using fresh culture media containing 0.004% w/v resazurin sodium salt  in 500 µL and incubated for 2 hours at 37°C. Next, 300 µL of reacted media was transferred to a new 96-well plate (100 µL in each well) and read on a Synergy 2 plate reader  at 600/25 nm, normalized with the cell-free media. Four samples per group were tested and compared to those of TCP and casted Ti disc controls.The expression of aldehyde dehydrogenase (ALDH3A1), integrin β1, α-smooth muscle actin (α-SMA), collagen I, and keratocan by HCFs cultured on Ti-(p)PMMA was determined by fluorescence immunocytochemistry after 6 or 45 days of cell culture. At the end of each culture period, the discs were removed from the media, rinsed with PBS, and fixed in 4% paraformaldehyde. Cells were permeabilized with 0.25% Triton X-100 and blocked with 5% fetal bovine serum in 0.05% Tween-20 in PBS. Cells were incubated with the following primary antibodies overnight at 4°C in humidified conditions: (1) mouse monoclonal antibody against ALDH3A1 ; (2) rabbit polyclonal antibody against integrin β1 ; (3) mouse monoclonal antibody against α-SMA ; (4) mouse monoclonal antibody against collagen I ; and (5) goat polyclonal antibody against keratocan . The specimens were incubated with specific secondary antibodies—Goat Anti-Mouse IgG H&L (FITC)  or Goat Anti-Rabbit IgG H&L (FITC) —for 1 hour at room temperature, washed with PBS, and mounted in VectaShield mounting media containing 4′,6-diamidino-2-phenylindole . They were then imaged using the Zeiss Axio Observer Z1 inverted fluorescent microscope.,HCFs were cultured as above, and after 45 days the total amount of collagen deposition per group PMMA, and Ti) was quantified using a total collagen assay kit  according to the manufacturer's protocol.Human blood was drawn from healthy volunteers into Vacutainer tubes  containing a specific thrombin inhibitor, lepirudin , at a final concentration of 50 µg/mL. All experimental protocols were approved by the ethics committee at Oslo University Hospital (REK SØR S-04114), and the all methods were carried out in accordance with relevant guidelines and regulations, conforming to the tenets of the Declaration of Helsinki. Informed written consent was obtained from each donor.,For each set of experiments, 300 µL of the blood was aliquoted into four 1.8-mL round-bottom sterile polypropylene cryogenic vials (Thermo Fisher Scientific). In one vial, ethylenediaminetetraacetic acid (EDTA) was added immediately at a 10-mM final concentration, thereby stopping further complement activation, in order to measure the complement activation status at time zero (T0). The other three vials containing whole blood were incubated at 37°C as follows. PMMA discs (8-mm diameter) were placed in one vial and Ti-(p)PMMA discs in another. The third vial contained only whole blood. After incubation, EDTA was added to stop further complement activation, and plasma was separated for preservation. The incubated whole blood without an immersed specimen served as a negative control for complement activation. Three sets of experiments were carried out with samples collected after 60, 90, and 120 minutes of incubation. Five independent sets of experiments were performed at 30 minutes of incubation. Collected plasma was preserved at –70°C. Complement activation was assessed by measuring soluble C3bc fragments and the soluble terminal complement complex (TCC) sC5b-9 and using enzyme-linked immunosorbent assays as described previously.t-test (two-tailed) was used to compare resolution and imputed visual acuity between groups (Ti-coated and non-coated BKs). A value of P < 0.05 was considered statistically significant. Prism 8.3.0  was used to analyze the data.One-way analysis of variance (ANOVA) with Tukey comparison test was used to compare surface roughness, contact angle, adhesion strength, scratch hardness, viability, metabolic activity, collagen content, and TCC among groups. Student's P = 0.00002 and P = 0.00034, respectively). Compared to all other groups, casted Ti showed an even higher roughness with parallel grooves (RMS = 102.22 ± 24.07 nm) PMMA, and Ti-(p)PMMA, with casted Ti serving as a control, was studied by AFM a–e. AFM 4.07 nm) d. The thP < 0.05) among all groups.The Ti coating surface energy and hydrophilicity were assessed using water contact angle measurements. The contact angles for PMMA, (p)PMMA, and Ti-(p)PMMA were 74.5° ± 3.2°, 56.9° ± 2.4°, and 42.6° ± 3.9°, respectively, as compared to casted Ti (50.4° ± 5.2°) f. ANOVA XPS spectra of PMMA discs (PMMA and Ti-(p)PMMA) are shown in P = 0.94)  c. All th = 0.94) a, 3d.P = 0.00002), as illustrated in P = 0.88).The adhesion of sputtered Ti to PMMA was evaluated using a pull-off adhesion test.P = 0.001) as shown in P = 0.998).The scratch load of Ti-(p)PMMA was significantly higher than that of Ti-PMMA  (P < 0.00001). HCFs cultured on Ti-(p)PMMA showed metabolic activity similar to HCFs on casted Ti and TCP controls  ; however, those seeded on the Ti-(p)PMMA surface exhibited greater confluency (59.3% ± 1.8%) compared to PMMA (18.2% ± 2.1%) after 7 days of cell culture. HCFs cultured on Ti-(p)PMMA demonstrated cell confluency similar to that of casted Ti and TCP controls  a, 4b. HCctively) a. The alctively) c.Further evaluation of the biological effects of Ti-(p)PMMA on cultured HCFs was performed using immunocytochemistry for ALDH3A1 (marker of keratocyte phenotype), integrin β1 (adhesion), and α-SMA (marker of fibroblast phenotype) a, 6b aftP = 0.00024) PMMA was higher b compare0.00024) c.n = 3) to both PMMA and Ti-(p)PMMA (n = 5), the level of TCC increased significantly (0.87 ± 0.38 CAU/mL) compared to the negative control . Complement activation (as measured by TCC) by Ti (p)PMMA was not significantly different (0.69 ± 0.34) compared to the negative control (P = 0.29). There was no significant difference in TCC levels between PMMA and Ti-(p)PMMA (P = 0.64) (Complement activation by PMMA and Ti-(p)PMMA was assessed for two critical nodes in the complement activation system. Levels of C3bc and soluble TCC increased over time with exposure of whole blood (-(p)PMMA a, 7b. Af = 0.64) c.The current study demonstrates that magnetron sputtering of Ti onto the stem of BKs is feasible and provides a mechanically robust and biocompatible surface. Our findings suggest that Ti sputtering on the BK stem may help corneal tissue adhesion and have the potential to improve clinical outcomes in patients with an implanted BK device.,,Surface properties of medical implants have been shown to significantly impact subsequent cellular behavior and function.P = 0.00002). For context, reported mean eyelid pressures based on three models of whole cell, imprint width, and Marx's line are 0.00051 ± 0.00001 MPa, 0.0011 ± 0.0004 MPa, and 0.0073 ± 0.0035 MPa, respectively, which are three to four orders of magnitude less than the adhesion strength of Ti-(p)PMMA.Coating of the PMMA surface must have sufficient mechanical stability to withstand cleaning, sterilization, and implantation of the device. Magnetron sputtered Ti film on PMMA has been explored previously,2/HAp dip-coatedScratch testing further demonstrated the stability of the sputtered Ti film and showed scratch resistance properties similar to those of casted Ti. In contrast, polydopamine,Migration of corneal stromal cells from the host corneal tissue to the surface of the BK PMMA stem and subsequent proliferation and production of ECM are desirable for adhesion of corneal tissue to the stem in order to prevent gaps between the BK and the donor cornea that would otherwise permit ingress of microbes. Therefore, it is important to determine the biological interaction of HCFs with coated PMMA surfaces. It has been shown that Ti, especially with low roughness, displays enhanced biocompatibility with respect to corneal cells.,,,Our study also found that HCFs seeded on a Ti-(p)PMMA surface expressed ALDH3A1, a keratocyte specific marker. In comparison, ALDH3A1 expression on untreated PMMA was reduced. This suggests that Ti-(p)PMMA is more permissive of a physiologically normal keratocyte phenotype.The complement system triggers inflammatory responses against a foreign body through a sequential enzyme cascade.Corneal fibroblast interactions and behaviors  under in vitro conditions might not reflect those under in vivo conditions. The chemical composition, local biomechanics, nanoarchitecture of the surrounding matrix, spatial arrangement of cells with respect to their neighbors, and physiological communication between tissues and organs all orchestrate cell behavior under in vivo conditions, and these relationships are absent in vitro.In conclusion, a Ti sputtered coating conferred favorable mechanical and biological properties to the BK PMMA stem. Ti sputtering of PMMA increased cell adhesion, proliferation, and collagen deposition and permitted maintenance of a more normal corneal stromal cell phenotype. These findings suggest that Ti sputtering may improve corneal tissue adherence to the BK stem and improve long-term clinical outcomes for recipients of the device.


Gliomas represent the largest class of primary central nervous system neoplasms, many subtypes of which exhibit poor prognoses. Surgery followed by radiotherapy and chemotherapy has been used as a standard strategy but yielded unsatisfactory improvements in patient survival outcomes. The S-phase kinase protein 2 (Skp2), a critical component of the E3-ligase SCF complex, has been documented in tumorigenesis in various cancer types but its role in glioma has yet to be fully clarified. In this study, we investigated the function of Skp2 in the proliferation, stem cell maintenance, and drug sensitivity to temozolomide (TMZ) of glioma.To investigate the role of Skp2 in the prognosis of patients with glioma, we first analyzed data in databases TCGA and GTEx. To further clarify the effect of Skp2 on glioma cell proliferation, we suppressed its level in glioblastoma (GBM) cell lines through knockdown and small molecule inhibitors (lovastatin and SZL-P1-41). We then detected cell growth, colony formation, sphere formation, drug sensitivity, and in vivo tumor formation in xenograft mice model.Skp2 mRNA level was higher in both low-grade glioma and GBM than normal brain tissues. The knockdown of Skp2 increased cell sensitivity to TMZ, decreased cell proliferation and tumorigenesis. In addition, Skp2 level was found increased upon stem cells enriching, while the knockdown of Skp2 led to reduced sphere numbers. Downregulation of Skp2 also induced senescence. Repurposing of lovastatin and novel compound SZL-P1-41 suppressed Skp2 effectively, and enhanced glioma cell sensitivity to TMZ in vitro and in vivo.Our data demonstrated that Skp2 modulated glioma cell proliferation in vitro and in vivo, stem cell maintenance, and cell sensitivity to TMZ, which indicated that Skp2 could be a potential target for long-term treatment. Gliomas represent the largest class of primary central nervous system neoplasms, many subtypes of which exhibit poor prognoses. Even with aggressive treatment strategies, i.e. surgery followed by irradiation and chemotherapy, the prognoses of diffuse glioma patients remained unsatisfactory . EncouraCip1/Waf1 and p27Kip1 [Cip1/Waf1 is a broad-acting cyclin-dependent kinase inhibitor, and its stability is essential for proper cell cycle progression and cell fate decisions. An increase in p27Kip1 has been found to significantly reduce Mixed-Lineage Leukemia self-renewal ability, promote the monocytic differentiation of leukemic blasts, and induce cell death [To this end, the S-phase kinase-associated protein 2 (Skp2) has been identified as a potential prospect worth investigating. It is a member of the F-box protein family and forms the Skp-Cullin-F-box complex (SCF complex) with Skp1, Cullin-1, and Rbx1. The SCF complex has been showed to trigger the ubiquitination and degradation of downstream proteins, and Skp2 recognizes substrates for proteasome degradation [ p27Kip1 , 4. p21Cll death . Skp2 hall death –11. In hAlthough it was reported that Skp2 could promote cell growth, migration, and invasion in glioma cells, the detailed function of Skp2 remains unclear . In this2. The astrocytes were maintained in Astrocyte Medium  supplemented with FBS, astrocyte growth supplement and penicillin/streptomycin . The glioma stem-like cells were cultured in conditional medium  supplemented with B27 (Thermo Fisher Scientific), epidermal growth factor (EGF) , basic fibroblast growth factor (bFGF) , and 1% penicillin/streptomycin.Cell lines U87, U118, U373, U343 and 293T were maintained from the State Key Laboratory of Oncology in South China. A172, U138, LNZ308 and normal astrocyte cell line (Ast) were obtained from Dr. Shing-shun Tony To, Department of Health Technology and Informatics, The Hong Kong Polytechnic University. The WHO classification and IDH mutation status of glioma cell lines applied in our study was listed in Additional file http://gepia.cancer-pku.cn/index.html) [We applied Gene Expression Profiling Interactive Analysis (GEPIA) to investigate the expression level of Skp2 in 24 tumor types and differential expression in GBM and low-grade glioma (LGG) compared with normal brain tissues. GEPIA is an interactive web application for gene expression analysis based on 9736 tumors and 8587 normal samples from the TCGA and the GTEx databases, using the output of a standard processing pipeline for RNA sequencing data . GEPIA iex.html) . We alsoThe preparation of viral particles was performed as in our previous publication . BrieflyCip1/Waf1 , anti-β-catenin , β-tubulin (Proteintech), and β-actin , and rabbit anti-p27Kip1 . The images were captured for further analysis on a BIO-RAD ChemiDoc Imaging System .Western blot analyses were performed as described previously , 17. TheCells were harvested, fixed with 70% ethanol overnight at 4 °C, and stained with propidium iodide  in the dark for 1 h. Cell suspensions were subjected to flow cytometry with an ACEA NovoCyte system . Cell cycle distribution was analyzed with 100,000 events for each sample.Flow cytometry analysis was used to detected apoptotic cell death. Briefly, cell suspensions were blocked with 10% BSA for 10 min. Apoptosis were detected using Annexin V–FITC/PI detection kit . Flow cytometry analysis was performed using a CytoFLEX  flow cytometer equipped with CytExpert software with 20,000 events recorded for each sample.Cells were quantified and seeded into 96-well plates. Cell viability was detected with a CCK8 kit  for 6 continuously days. Growth curves were then made according to the observation density (OD) values.24H36O5, 404.54 g/mol, Merck Millipore, Darmstadt, Hessen, Germany) and SZL-P1-41  were used for combination treatment with TMZ . Lovastatin (25 mg) was dissolved in DMSO (6.18 ml) to get the stock solution (10 mmol/l), which was further diluted into 1 μmol/l with culture medium for further application. Similarly, the stock solution of SZL P1-41 (5 mmol/l) was prepared by dissolving 10 mg in 4.756 ml of DMSO, which was further diluted into 1 mmol/l with culture medium for further use. Cells were counted and seeded into 96-well plates on day 1, and pretreated with lovastatin or SZL-P1-41 on day 2. The final concentration of lovastatin and SZL P1-41 applied to cells were 10 nmol/l and 20 μmol/l respectively. TMZ was applied on day 3 for 72 h (together with lovastatin or SZL-P1-41) and the cell viability was detected. The survival rate was drawn according to the OD and compared to nontreated cells. The half-maximal inhibitory concentration (IC50) was calculated by GraphPad Prism 5 . All experiments were repeated at least three times, and each concentration had four independent repeats.Cells viability was tested using the CCK8 assay after treatment. Lovastatin  for sphere formation assay in stem-like cell conditional medium, at a density of 1000 cells per well as reported . After cThe assay was performed as reported previously . Briefly7 cells was subcutaneously injected into the back of nude mice. The resulting tumor size was measured with a Vernier caliper every 3 days and tumor weight was determined immediately after separated from mice. Tumor-bearing mice were treated with TMZ when tumor volume reached 80 mm3. The mice were observed for over 4 weeks and sacrificed before dying of tumor burden.The in vivo animal work followed previously published protocol . Four-weData obtained from both in vitro and in vivo are expressed as the mean ± standard error. p < 0.05 was considered statistically significant. Statistical analysis was performed with Graph Pad Prism 5.0.mut and Skp2low  patients since there was no 1p19q co-deletion in the IDHwt LGG patients (n = 94). We found that the combination of 1p19q non-co-deletion and Skp2high contributed to the poor prognoses in IDHmut LGG compared with other three groups, 1p19q co-deletion/Skp2low, 1p19q co-deletion/Skp2high and 1p19q non-co-deletion/Skp2low  was found stronger than in AST cells -goce models , 31–34. e models . Our stuCurrently, drug repurposing has become of increasing interest and evolved as a promising field in cancer treatment , 36. LovIn this study, we demonstrated that Skp2 increased with glioma grade and predicted a poor prognosis in diffuse glioma as well as LGG. We also demonstrated that the downregulation of Skp2 led to the retardation of cell proliferation, increased TMZ sensitivity, reduced the sphere formation ability of glioma stem-like cells, and enhanced cellular senescence. Furthermore, two small molecules (lovastatin and SZL-P1-41) could target Skp2 to improve TMZ efficiency in vitro and in xenograft mouse model in vivo. Our results therefore reveal the important role of Skp2 in glioma tumorigenesis, targeting Skp2 could potentially improve the therapeutic efficiency of glioma patients.Additional file 1: Table S1. WHO classification and IDH mutation status of glioma cell lines applied in our study. Table S2. Clinical information of the LGG and GBM cohort included for our OS analysis from TCGA database.Additional file 2: Figure S1. a–c The role of Skp2 expression in the OS of patients with IDH1wt GBM, IDHwt LGG and even IDHmut LGG was analyzed. d, e The role of Skp2 expression in the OS of IDHmut LGG patients with or without 1p19q co-deletion was analyzed. Figure S2. Cell cycle cascade rate were detected in three cell lines, U87, U138 and LNZ308, upon LV, TMZ or combined treatments. With regard to the strong effect of TMZ in glioma cells, we chose 10 nM as the combination concentration of lovastatin in our study. Lovastatin caused a little bit of G2/M arrest in U87 and LNZ308, but not in U138 cell. The G1 phase cell numbers were enhanced in U138 and LNZ308 cells upon lovastatin treatment. Lovastatin did not further induce G2/M arrest when combined with TMZ in U87 and U138. However, in LNZ308 cells, lovastatin promoted G2/M arrest upon TMZ treatments dramatically. . Figure S3. Lovastatin barely induced cell apoptosis in U87 and U138 cells but promoted cell apoptosis in LNZ308 cells markedly at 10 nM. When combined with TMZ, lovastatin did not promote the apoptotic cell rates in U87, and even antagonized the apoptosis induction effect in U138 and LNZ308 cells. .


Background: To assess the role of nodal involvement in stage III renal cell carcinoma (RCC) according to the American Joint Committee on Cancer (AJCC) 8th staging system. We compared the survival outcomes of RCC patients with pT1−3N1M0 disease and those with pT3N0M0 or stage IV (stratified as pT4NanyM0 and pTanyNanyM1) disease in a large population-based cohort.Methods: A cohort of 3,112 eligible patients with RCC was identified from the Surveillance, Epidemiology, and End Results (SEER) database, registered between January 2004 and December 2015. Kaplan-Meier and Cox proportional hazards models were used to evaluate the overall survival (OS), and cancer-specific survival (CSS). The prognostic value of the modified stage for pT1−3N1M0 disease was assessed by nomogram-based analyses. Propensity score matching (PSM) was used to adjust for potential baseline confounding.Results: Patients with pT1−3N1M0 disease showed similar survival outcomes  to pT4NanyM0 patients, whereas the significantly better survival outcome was found for pT3N0M0 patients. After PSM, comparable survival rates were observed between pT1−3N1M0 group and pT4NanyM0 group, which were still significantly worse than the survival of pT3N0M0 patients. The modified stage IIIA (pT3N0M0), IIIB , and IV (pTanyNanyM1) showed higher predictive accuracy than AJCC stage system in the nomogram-based analyses .Conclusions: The pT1−3N1M0 RCC might be reclassified as stage IIIB together with pT4NanyM0 disease for better prediction of prognosis, further examination and validation are warranted. The study cohort was then limited to patients with stage III disease or pTanyNanyM1 and pT4NanyM0 disease according to the AJCC 8th Staging Manual using the collaborative stage information . The eligible criteria included in the study were participants with one primary only, pathologically confirmed RCC, and contained complete data of age, race, gender, surgery records, pathological information, and with active follow up; and all patients underwent radical or partial nephrectomy with LN dissection which guaranteed the specimen and harvested LNs were sent for pathology in SEER , sex , race , tumor site , and histological subtypes . Grade in SEER is coded according to the ICD-O-3. The number of examined and positive LNs is queried from collaborative stage information and presented as a continuous variable. Since information of invasion beyond capsule, fuhrman nuclear grade, and sarcomatoid features are only applicable after 2010, these covariates are not examined in χ2-test .PSM is a tool to reduce selection bias in observational studies . PSM wit2-test were performed to compare the distribution of baseline characteristics. The survival curves were estimated by the Kaplan-Meier method and compared by the log-rank test. The univariate and multivariate analyses and hazard ratios (HR) were evaluated by Cox proportional hazards regression model to find its independent prognostic risks. Significant variables in univariate analysis were entered into a multivariate model, and variables that remained significant were further entered into the final multivariate regression model. P ≤ 0.05 (2-sided probability) was considered statistically significant.Overall survival (OS) was defined as the time from diagnosis to death from any cause; cancer-specific survival (CSS) was defined as the time from diagnosis to death related to RCC. Frequencies and proportions, as well as means were reported for categorical and continuous variables, respectively. General linear models or χrms packages in R , pT1−3N1M0 group (n = 446), pT4NanyM0 group (n = 105), and pTanyNanyM1 group . 1−3N1M0 group , patients in pT1−3N1M0 group are more likely to have undifferentiated tumors  and papillary adenocarcinoma  than those in pT3N0M0 group. After PSM, all variables between the study groups were balanced, except for those variables that were only applicable after 2010.Between January 2004 and December 2015, 3,112 eligible patients were identified and categorized into pTP < 0.001), higher fuhrman grade (P < 0.001), presence of sarcomatoid features (P < 0.001), and presence of node disease (P < 0.001) were associated with remarkably worse OS and CSS among stage III RCC patients before matching. After matching, similar outcomes were observed for stage III RCC patients (P = 0.002) and CSS (P = 0.001) compared to patients with clear cell type RCC, which indicated the potential survival benefit of chrompohobe type RCC  and after matching .patients . Moreovetype RCC . In the 3N0M0 group , whereas the similar survival was found for pT1−3N1M0 group and pT4NanyM0 group . Moreover, significantly worse survival  were found for pTanyNanyM1 group  compared with those of pT1−3N1M0 group  compared to the AJCC 8th stage group in the primary cohort as well as the validation cohort , which indicated the higher discriminatory power of the modified stage.Given the above results, pT3N0M0 disease could be reclassified as stage IIIA, while pT1−3N1M0 RCC together with pT4NanyM0 disease could be classified as stage IIIB for better prediction of prognosis.To the best of our knowledge, the present study is the first population-based analysis using the PSM method to assess the impact of LN involvement on survival in stage III RCC patients. In the current study, LN involvement, in general, was associated with remarkably poor survival in patients with stage III disease in the AJCC 8th staging system in both multivariate regression and PSM analyses. We found that RCC patients with pTP < 0.001). Srivastava et al. found the consistent outcomes for node-negative and positive group in their NCDB cohort  (P < 0.001) , and thus reinforcing these findings.LN involvement in RCC patients has been decreasing over years, from 30% in historical series to approximately 6%-20% in more recent studies , 26–28.  months) . A more < 0.001) . Compara0M1 disease and 236 (26%) with pN+M0 disease, among the 900 study patients (P = 0.59). MDACC study compared the pT1−3N1M0 disease with pT1−3N0M1 disease in OS and CSS, and found that pT1−3N1M0 disease was not different in both OS  and CSS  relative to pT1−3N0M1 diseases . Given the better survival of T3N0M0 disease in the current study, these controversial findings indicated that T3N0M0 may not significantly benefit from adjuvant therapy similar to T1−3N1M0 and T4NanyM0 disease. The poor survival observed for pT1−3N1M0 and pT4NanyM0 disease in our study might partly support the necessity of adjuvant therapy. Thus, it might be appropriate to regroup the pT1−3N1M0 disease together with pT4NanyM0 disease for better guiding adjuvant therapy.Pathologic staging of RCC is essential for guiding clinical treatment decision-making process and selecting patients for potential adjuvant therapy . High-ri4 disease for the similar CSS between ipsilateral adrenal invasion and pT4 disease  and classified as stage IIIB together with pT4NanyM0 disease. Further researches and independent cohort validations are warranted to address the heterogeneity of the stage III RCCs.In conclusion, the present study suggested that the current AJCC 8th staging system for stage III RCC should be further discussed and validated for the better prediction of prognosis. If validated, patients with pTThe datasets generated for this study are available on request to the corresponding author.Patient consents were not required because the study is a retrospective database research in nature, there was no direct patient contact. Institutional Review Board approval was not required according to our institution policy.ZF: guarantor of the article. ZF and JH: conception/design. JH, QL, PL, SW, YQ, and RZ: collection and/or assembly of data. JH, PL, QL, RZ, SW, YQ, and QS: data analysis and interpretation. JH, QL, PL, SW, RZ, YQ, QS, and ZF: manuscript writing. ZF, JH, PL, SW, QL, RZ, YQ, and QS: final approval of manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Sargassum fusiforme sludge. When the strain was grown on a plate containing sodium alginate, the transparent ring diameter (D) was 2.2 cm and the ratio (D/d) of transparent ring diameter to colony diameter (d) was 8.8. After 36 h in culture at a temperature of 28 °C shaken at 150 r/min, the enzymatic activity of the fermentation supernatant reached 160 U/mL, and the enzymatic activity of the bacterial precipitate harvested was 2,645 U/mL. The strain was named Cobetia sp. cqz5-12. Its genome is circular in shape, 4,209,007 bp in size, with a 62.36% GC content. It contains 3,498 predicted coding genes, 72 tRNA genes, and 21 rRNA genes. The functional annotations for the coding genes demonstrated that there were 181 coding genes in the genome related to carbohydrate transport and metabolism and 699 coding genes with unknown functions. Three putative coding genes, alg2107, alg2108 and alg2112, related to alginate degradation were identified by analyzing the carbohydrate active enzyme (CAZy) database. Moreover, proteins Alg2107 and Alg2112 were successfully expressed and exhibited alginate lyase activity.Alginate-degrading bacteria or alginate lyases can be used to oligomerize alginate. In this study, an alginate-degrading bacterium with high alginolytic activity was successfully screened by using  Gigantophtha, Charcot algae, Laminaria japonica, and Sargassum fusiformis. It is formed by the random combination of beta-d-1,4-mannosuronic acid and alpha-l-1,4-gulosuronic acid2. Because of its high degree of polymerization, high molecular weight, and high viscosity, it not easily absorbed and utilized by the human body, which limits its potential applications3. The oligomerization of alginate can widen the scope of its potential applications and results in more biological activities. The oligomeric alginate is called alginate oligosaccharide (AOS). AOS, a kind of functional oligosaccharide, exhibits a variety of properties such as increasing fruit quality4, and antioxidative5, anticoagulant6, and antineoplastic7 potential. Moreover, AOS has broad application prospects in the fields of pharmaceuticals, functional food, and green agriculture. Nowadays, there are many ways to achieve alginate oligomerization. At present, it is recognized that the environmentally friendly and mildly efficient method of alginate oligomerization is to use alginate-degrading microorganisms or alginate lyase8 extracted from microorganisms. Until now, scientists have isolated several strains of alginate-degrading microorganisms, including Cobetia sp. NAP19, Microbulbifer mangrovi DD-13T10, Vibrio weizhoudaoensis M01011, Isoptericola halotolerans NJ-0512, V. splendidus 12B0113, Sphingomonas sp. A114, Pseudoalteromonas sp. CY2415, etc. Some alginate lyase enzymes have also been isolated and purified from these strains15.Alginate is a kind of water-soluble acidic polysaccharide found on the cell walls of algae, such as Cobetia marina JCM 21022T using high-throughput sequencing and determined the taxonomic status and functional differences between this strain and other strains of related families and genera by comparative genomics16. Md. Imran et al. also obtained the complete genomic information of Microbulbifer mangrovi DD-13T using high-throughput sequencing, and identified a series of genes related to polysaccharide degradation through functional annotation12. Thereafter, the polysaccharide substrate spectrum of the strain was verified based on genetic information. By considering both the crude enzyme experiments and the whole genome sequence information, researchers determined that sodium alginate can be thoroughly degraded by Algibacter alginolytica sp. novHZ22, and proposed that the structure and mechanism of polysaccharide utilization loci (PUL) be the goal of further research17.With the improvement of sequencing technology and the dramatic reduction in sequencing costs, the basic information about a target strain can be quickly obtained by using high-throughput technology to determine its whole genome sequence. The sequence of relevant coding genes can also be quickly obtained by means of gene annotation. Tang et al. obtained the complete genomic sequence information of Cobetia sp. cqz5-12 was isolated from Sargassum fusiformis sludge taken from the Dongtou sea area, Wenzhou, Zhejiang Province, China. In order to obtain basic information about the strain quickly, whole genome sequencing of the strain was performed by using the Illumina Miseq + Pacbio sequencing platform. Through bioinformatics analysis, the complete genomic information about the strain was obtained, from which the coding gene sequences potentially related to alginate degradation were obtained and attempts were made to express these sequences. The identification, genome sequencing, and target gene expression of Cobetia sp. cqz5-12 aid in the better understanding of the specific alginolytic properties of this strain. Further research can be performed using this data with the goals of understanding the degrading mechanism at the molecular level and, in turn, improving its alginolytic efficiency.In order to select the most efficient alginate-degrading microorganism or alginate lyase for use in the industrial production of AOS, we needed to modify the present alginate-degrading microbe or alginate lyase, or else isolate a new, more efficient alginate-degrading microorganism, and purify a new alginate lyase from it. For this purpose, the strain Sargassum fusiformis. When a colony of this strain formed on a solid plate containing sodium alginate, 1% calcium chloride was used to soak the colony for 30 min, and then a transparent circle could be clearly observed around the colony , moist, and smooth  were obtained. The total length of the CDS was 3,525,474 bp, accounting for 83.76% of the total genome length. The average length of the CDS was 1,007.85 bp, the average GC content of the CDS was 63.50%, and the maximum CDS length was 20,397 bp. By predicting non-coding RNA, 21 rRNAs (5s + 16s + 23s), 72 tRNAs, and 114 other ncRNAs were obtained, accounting for 0.7534%, 0.1337% and 0.7522% of the total length of the genome sequence, respectively  revealed that 236 protein-coding genes contained signal peptide sequences, accounting for 6.75% of the total CDS, and there was at least one transmembrane helical region in 872 CDS, accounting for 24.9% of the total CDS. There were sequences of secretory proteins in 78 coding genes, accounting for 4.95% of the total CDS.19. Therefore, about 20% of the CDS of this strain may encode new proteins.Sequence blast of the CDS and functional annotation analysis of CDS were performed. The analysis results showed that 229 CDS were related to energy production and conversion, 181 were related to carbohydrate transport and metabolism, 169 were related to translation, ribosomal structure and biogenesis, 224 were related to transcription, 200 were related to cell wall/membrane/envelope biogenesis, 29 were associated with cell motility, 230 were associated with inorganic transport and metabolism, 58 were associated with secondary metabolites biosynthesis, transport and catabolism, 138 were associated with signal transduction mechanisms, 57 were related to intracellular trafficking, secretion and vesicular transport, and the function of 699 CDS were unknown , 17 glycoside hydrolases (GHs), 39 glycosyltransferases (GTs), 14 carbohydrate esterases (CEs), seven carbohydrate binding modules (CBMs), and 11 Auxiliary Activities (AAs).The analysis of carbohydrate-active enzymes (CAZy) of the CDS was performed by using the CAZy database and hmmscan software alg1771, alg2107, alg2108, and alg2112, respectively. alg1771, 1,272 bp in length, was annotated as the transporter TolB; alg2107, alg2108 and alg2112, the lengths of which were 2,142, 2,160 and 915 bp, respectively, were annotated as hypothetical proteins, oligosaccharide lyases and polysaccharide lyases, respectively. Furthermore, the enzymatic activities of the fermentation supernatant and the cell disruption supernatant of Cobetia sp. cqz5-12 were 160 and 2,645 U/mL respectively, which indicated that the alginate lyase produced by the bacteria was mostly retained inside the cell. According to this lyase assay result, we thought that alg1771 might play a minor role in the secretion of alginate lyase. Based on these data, alg2107, alg2108, and alg2112 were selected for further experimental verification.Based on the result of CAZy analysis, the four polysaccharide lyases are encoded by genes numbered 1771, 2107, 2108 and 2112. The genes were named The SDS-PAGE analysis of the crude protein extract showed that Alg2107 and Alg2112 successfully appeared in the theoretically expected position, while Alg2108 did not Fig. . The theVibrio sp. QD-5 isolated from rotting kelp22 and alginate lysase AlgA from the marine bacterium Bacillus sp. Alg0723. Cobetia sp. cqz5-12, the research topic of this study, was isolated from rotten Sargassum fusiformis. After incubation in a shaking culture containing sodium alginate, the sodium alginate lyase activity of the culture supernatant fluid was 160 U/mL, significantly lower than the value of 510 U/mL found in the marine bacterium Bacillus sp. Alg0723 reported previously. However, the enzymatic activity of cell disruption supernatant of Cobetia sp. cqz5-12 reached 2,645 U/mL. Nevertheless, the enzymatic activity of the cell disruption supernatant of Bacillus sp. Alg07 has not been reported. Both the culture supernatant of Cobetia sp. cqz5-12 and the cell disruption supernatant of Cobetia sp. cqz5-12 had alginolytic activity, indicating that there may be more than one enzyme in strain cqz5-12 capable of degrading alginate. Some of the reported alginate lyases are intracellular enzymes22, which can be obtained from alginate-degrading bacteria by ultrasonication24 or purification after heterologous expression25. Others are extracellular enzymes29, which can be purified directly from the culture supernatant of the alginate-degrading bacteria, such as AlyV527. Therefore, we believe that strain cqz5-12 may produce multiple enzymes, both intracellular and extracellular, which can degrade alginate.Alginate is the main component on the cell wall of brown algae. At present, scientists have isolated and purified a large number of alginate-degrading marine microorganisms and alginate lyases from decaying algae, seawater, or seawater sediment, such as Cobetia sp. cqz5-12 was obtained. The genome size of Cobetia sp. cqz5-12 was 4,209,007 bp and its GC content was 62.36%. Compared with the most homologous strain, Cobetia Marina JCM 21022T12, the genome size and GC content of strain cqz5-12 were not significantly different from those of the strain JCM 21022T . However, the gene involved in alginate degradation may differ between strains cqz5-12 and JCM 21022T , close to 3,48810 CDS found in alginate-degrading bacterium Microbulbifer mangrove DD-13T, but higher than that of the alginate-degrading bacterium Algibacter alginolytica HZ22 17. Alginate-degrading bacterium Algibacter alginolytica HZ22 and Mirobulbifer mangrovi DD-13T contained 255 (18 PLs) and 461 CAZymes (17 PLs), respectively, while Cobetia sp. cqz5-12 only had 92 CAZymes (4 PLs), much fewer than those previously reported in other strains. Although strain cqz5-12 contained fewer CAZymes, two of the four PLs genes were annotated as enzymes associated with alginate lysis , respectively 2SO4, 30.0 g/L NaCl, 2.0 g/L K2HPO4, and 0.01 g/L FeSO4·7H2O at pH 7.0. The solid medium A was prepared by adding 1.8% agar powder to the above medium. The liquid fermentation medium was formulated as 6.0 g/L sodium alginate, 5.0 g/L tryptone, 2.5 g/L yeast powder, 25.0 g/L NaCl, 0.25 g/L KH2PO4, 0.5 g/LMgSO4·7H2O, and 0.05 g/L FeSO4·7H2O at pH 7.5. The formula of the seed liquid medium was the same as that of the liquid fermentation medium.The formulation of the selective liquid medium used in the strain-isolating experiment was: 5 g/L sodium alginate, 1.0 g/L MgSOEscherichia coli DH10B, E. coli BL21 (DE3) and pET29a, all came from our laboratory’s strain bank, except that the pMD 19-T vector was purchased from TaKaRa, and the degrading strain was isolated from rotting Sargassum fusiformis.The other strains and plasmids used in the experiment, including Sargassum fusiforme was cut into small pieces with sterile scissors, placed in 10 mL of selective liquid medium, and incubated in a shaking culture at 150 r/min and 28 °C. Twelve hours later, 1 mL of the bacterial culture solution was removed and added to 25 mL of fresh selective liquid culture medium. Incubation was continued with shaking at 150 r/min at 28 °C. After 48 h, 1 mL of the bacterial suspension was removed and diluted with sterile seawater adjusted to 10–4 gradient, then 100 μL of the dilution was applied to solid medium A. When a single colony developed, it was immediately transferred to a fresh solid medium A. After the colonies had grown out on the new plate, 1% calcium chloride solution was added and soaking took place for 30 min to observe whether transparent circles or white halos around the colonies were present. Single colonies within transparent circles or white halos were transferred to a new solid medium and incubation was continued. After the colonies grew out, a single colony was selected and inoculated into the seed liquid culture medium. Shaking culture proceeded overnight at 28 °C and 150 r/min. After harvesting the bacteria, the seed liquid was inoculated into 25 mL liquid fermentation medium, and cultured at 28 °C and 150 r/min for 36 h. The bacterial solution was centrifuged for 5 min at 4 °C and 12,000 r/min. The supernatant was removed and enzyme activity was determined. The strains with higher enzymatic activity were selected. The strains obtained were further screened by flask-shaking fermentation, and the strain with the highest alginate-degrading ability was finally selected.1 g naturally rotted E. coli DH10B. The positive transformants were selected and sent to a biotechnology company for sequencing. Blast analysis was carried out on the sequencing results, and a phylogenetic tree was constructed using MEGA 7.0 software.Gram staining and microscopic observation was carried out on the alginate-degrading strains. Meanwhile, genomic DNA was extracted. The 16S universal primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-TACGGCTACCTTGTTACGACTT-3′) were used in the PCR reaction, and the products of the PCR reaction were purified, inserted into a pMD-19T vector, and then transformed into 35. The whole genome sequencing project was carried out by Shanghai Personal Biotechnology Co., Ltd. The whole genome shotgun strategy was used to construct the library of different inserts. The libraries were sequenced separately by using next-generation sequencing (Illumina MiSeq sequencing platform), as well as third-generation single-molecule sequencing (PacBio sequencing platform). The off-line data were obtained after DNA extraction, purification, library building, and sequencing. Further, the data obtained using second-generation sequencing were filtered by the filter standard to obtain high-quality data37. The off-line data obtained from the third-generation sequencing were assembled using HGAP4 software39, then the contig sequences were obtained based on these analyses. The contig sequences were adjusted by high-quality data via Pion (version 1.22) software40, and then the adjusted sequences were spliced to obtain the complete sequence.Genomic DNA was extracted according to the method proposed by Marmur and Doty18. The predictions of tRNA genes, rRNA genes, and other non-coding RNAs genes were achieved by using tRNAscan-SE (version 1.3.1) software41, Barrnap (0.9-dev) (https://github.com/tseemann/barrnap), and the Rfam database, respectively42. Sequence blast of a protein coding sequence (CDS) was performed using diamond software (v0.9.10.111)43 and the NCBI nr database (release 2017.10.10). We also predicted signal peptide44, transmembrane helix45 and secretory protein encoding genes, and then performed the annotation of eggNOG 46, KEGG (Kyoto Encyclopedia of Genes and Genomes)47 and GO48. The CAZy (Carbohydrate-Active enZYmes Database) analysis21 was also carried out. The whole genome map was drawn via cgview49, and Photoshop CS was used to edit the map.The genes of the whole genome sequence were predicted by GeneMarkS (version 4.32 April 2015) softwarealg2107, alg2108 and alg2112 were obtained by PCR  vector and the recombinant expression vectors pET29a-Alg2107, pET29a-Alg2108, and pET29a-Alg2112 were obtained. Subsequently, the recombinant expression vectors were successfully transformed into the host strain E. coli BL21 (DE3) for protein expression, and the expression strains of E. coli BL-07, E. coli BL-08 and E. coli BL-12 were obtained.The gene fragments A single colony of each protein expression strain was inoculated into LB (Luria–Bertani) liquid medium containing kanamycin and incubated overnight at 37 °C with shaking. The overnight culture was transferred to LB liquid medium containing kanamycin and cultured at 37 °C in a concussive manner.600 value of the bacterial solution reached approximately 0.5, a final concentration of 0.2 mmol/L isopropyl beta-D-1-thiogalactopyran glycoside (IPTG) was added. Then the strains were cultured at 20 °C in a concussive manner for 24 h and harvested. The bacteria were precipitated by centrifugation at 8,000 r/min for 5 min. The bacterial precipitate was re-suspended in buffer A  and fragmented by ultrasound for 2 min. After 20 min of centrifugation at 4 °C and 12,000 r/min, the cell disruption supernatant was collected and used to for enzyme activity determination and further protein purification. The cell disruption supernatant was then purified through Ni–NTA chromatography, dialysis, and ultrafiltration.When the ODProtein concentrations were determined by Bradford method and protein electrophoresis was performed using the SDS-PAGE method.The 3,5-dinitrosalicylic acid (DNS) method was used to detect the amount of reducing sugar released by enzymatic reaction. Then, the enzymatic activities of the alginate-degrading proteins were calculated according to the increase in reducing sugar content. A suspension of 0.6% of the sodium alginate substrate solution was prepared in disodium hydrogen Phosphate-citric acid buffer at pH 7.4. 1 mL properly diluted crude extract or purified enzyme solution was mixed with 1 mL of the substrate solution in a colorimetric tube, and then incubated in a water bath at 40 °C for 20 min. After adding 1.5 mL DNS solutions, the sample was immersed in a boiling water bath for 5 min, then immediately cooled to room temperature. The absorbance at 540 nm was determined after setting the sample volume to 10 mL. In blank controls, 1 mL inactivated crude extract was added to the substrate.A unit of enzyme activity was defined as the amount of enzyme required to catalyze substrate to produce 1 μg of reducing sugar per minute. All enzymatic reactions were performed in three parallel experiments.Cobetia sp. cqz5-12 has been deposited at the DDBJ/EMBL/GenBank database under accession number CP044522.The complete genome sequence of Cobetia sp. cqz5-12 has been deposited in the China General Microbiological Culture Collection Center, and the collection number is CGMCC 1.17459.Supplementary file1 (PDF 676 kb)