Some pproliferation growth blockers are more Inhibit cancer cell proliferation to cause some side proliferarion and these Inhibit cancer cell proliferation sometimes be serious. Although considered one of the most chemosensitive solid tumors 2usually after a positive response, they often relapse and develop resistance to a broad spectrum of drugs. Int J Biol Biol Sci.

Inhibit cancer cell proliferation -

Quercetin suppresses NLRP3 inflammasome activation in epithelial cells triggered by Escherichia coli OH7. Article CAS Google Scholar. Li, Z. Kinases Involved in Both Autophagy and Mitosis. Vogt, P. Phosphatidylinositol 3-kinase: the oncoprotein.

Article MathSciNet PubMed CAS Google Scholar. Ouyang, Z. Zhang, H. Cytotoxic effects of procyanidins from Castanea mollissima Bl. shell on human hepatoma G2 cells in vitro. Zhou, M. Saiprasad, G.

Download references. This work was supported by the National Natural Science Foundation of China ; ; College of Pharmacy, 3rd Military Medical University, Chongqing, , China. You can also search for this author in PubMed Google Scholar. and J. conceived and designed the research study.

isolated and characterized the structures of flavonoids, Q. performed the homology modeling and molecular docking. wrote and revised the manuscript. and Q. participated in the experiments. performed the statistical analysis.

All authors read and approved the final manuscript. Correspondence to Ning Gao. Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4. Reprints and permissions.

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Sign up for the Nature Briefing: Cancer newsletter — what matters in cancer research, free to your inbox weekly. Skip to main content Thank you for visiting nature. nature scientific reports articles article. Download PDF. Subjects Breast cancer Pharmaceutics. Abstract Anticancer activities of flavonoids derived from Tephroseris kirilowii Turcz.

Introduction Traditional Chinese medicines have been recently recognized as a new source of anticancer drugs and neoadjuvant chemotherapy to enhance the efficacy of chemotherapy and to alleviate the side effects of cancer chemotherapy 1 , 2. Materials and Methods Isolation, purification and identification of flavonoids from Tephroseris kirilowii Turcz.

Holub Tephroseris kirilowii Turcz. Chemicals and antibodies AS S and nocodazole were purchased from Selleck Chemicals Shanghai, CA.

Cell culture MDA-MB, MCF-7, A, SMMC, Eca, HEB and MCFA cells were provided by the American Type Culture Collection ATCC, Manassas, VA. Cell cycle analysis Cells were harvested, washed twice with phosphate-buffered saline PBS and incubated with nuclei staining buffer 0.

Western blots Cells were processed for western blotting as described previously Molecular docking study The binding modes of three flavonoids IH, GN, and Aca with kinases were predicted by Surflex-Dock in SYBYL2.

PI3K kinase assay ADP-Glo luminescent assay for PI3Kγ was performed according to the standard protocols of Promega. Statistical analysis Statistical analysis was performed with SPSS 20 software SPSS, Chicago, Illinois, USA. Results Anticancer activity screening of flavonoid compounds from T.

kirilowii Eight flavonoid compounds were isolated for the first time from T. Figure 1. Full size image. Table 1 IC50 of Flavonoids-inhibited cell proliferation in a variety of cancer. Full size table. Figure 2. Figure 3. Figure 4. Figure 5. Figure 6. Figure 7. Discussion The ability of flavonoids to inhibit cell proliferation and induce apoptosis or autophagy in human cancer cells has stimulated intense interest in their potential as anti-cancer agents.

References Xu, H. Article PubMed PubMed Central CAS Google Scholar Liu, Z. PubMed Google Scholar Panche, A. Article PubMed PubMed Central CAS Google Scholar Luo, X.

Article PubMed CAS Google Scholar Cao, Y. Article PubMed CAS Google Scholar Ding, Y. Article PubMed CAS Google Scholar Dai, J. Article PubMed PubMed Central CAS Google Scholar Zhao, K.

Article PubMed PubMed Central CAS Google Scholar Yang, Y. PubMed Google Scholar Wang, Y. Article Google Scholar Liu, J. Article PubMed Google Scholar Efferth, T. Article PubMed CAS Google Scholar Chen, M.

Article PubMed Google Scholar Chen, G. Article PubMed PubMed Central ADS Google Scholar Prieto-Dominguez, N. Article PubMed PubMed Central CAS Google Scholar Bizzarri, R. Article PubMed CAS Google Scholar Ferreira, L. Article PubMed CAS Google Scholar de Ruyck, J. Article PubMed Google Scholar Zheng, H.

Article PubMed PubMed Central Google Scholar Sui, X. Article PubMed CAS Google Scholar Azzi, J. Article PubMed PubMed Central CAS Google Scholar Ebrahimi, S. Article PubMed CAS Google Scholar Li, C.

Article PubMed CAS Google Scholar Nasr Bouzaiene, N. Article PubMed CAS Google Scholar Hsu, Y. Article PubMed CAS Google Scholar Singh, R. Article PubMed CAS Google Scholar Kishimoto, T. Article PubMed PubMed Central CAS Google Scholar Ryu, H. Article PubMed CAS Google Scholar Wang, S.

Article PubMed CAS Google Scholar Wu, L. Article PubMed PubMed Central CAS Google Scholar Xue, Y. Article CAS Google Scholar Li, Z. Article MathSciNet PubMed CAS Google Scholar Ouyang, Z. Article PubMed PubMed Central Google Scholar Zhang, H.

Article CAS Google Scholar Zhou, M. Article PubMed CAS Google Scholar Download references. Author information Author notes Hong-Wei Zhang and Jin-Jiao Hu contributed equally to this work. View author publications. Ethics declarations Competing Interests The authors declare no competing interests.

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Dataset 1. Rights and permissions Open Access This article is licensed under a Creative Commons Attribution 4. About this article. Cite this article Zhang, HW.

Copy to clipboard. This article is cited by Utilizing X-ray fluorescence XRF method to evaluate the content of metal elements in soil and their effects on the total phenolic and flavonoid contents of some medicinal plants Hien Minh Nguyen Ngan Thi Kim Huynh Thi Tan Pham Environmental Monitoring and Assessment Investigation of the Therapeutic Effects of Palbociclib Conjugated Magnetic Nanoparticles on Different Types of Breast Cancer Cell Lines Maryam Parsian Pelin Mutlu Ufuk Gunduz Cellular and Molecular Bioengineering Kaempferide exhibits an anticancer effect against hepatocellular carcinoma in vitro and in vivo Gopika Chandrababu Merlin Varkey Lekshmi R.

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To study cell surface phosphatidylserine exposure, an early marker of apoptotic cell death, MCF7, MCF7 ADR and MDA-MB cells were treated with 5 μM lauryl gallate for 0.

MCF7 C4 and E6, Bcl-2overexpressing MCF7 ADR clone MAB25 and its control transfectant clone MAN9 , MDA-MB clones 1 and 8, expressing temperature-sensitive p53, were treated with 5 μM lauryl gallate for 6 and 24 h. Cells were seeded in complete medium 1. After 24 h, samples were treated with 5 μM lauryl gallate for 48 h.

After this treatment, both detached and adherent cells were collected and washed with PBS. The pellet was fixed with 2. Samples were embedded in Epon resin Electron Microscopy Science, Fort Washington, PA. Ultrathin sections, obtained with a LKB ultramicrotome LKB, Bromma, Sweden , were stained with uranyl acetate and lead citrate and examined with a Philips S electron microscope Philips Electron Optics B.

Cells were treated with 5 μM lauryl gallate for 6 and 24 h. Finally, cells were washed and resuspended in cold PBS for flow cytometric analysis. JC-1 accumulates into the mitochondria as both monomers responsible for green fluorescence emission, FL1 and aggregates red fluorescence emission, FL2 depending on MMP.

Depolarization of the mitochondrial membrane is represented by an increase of FL1 and a decrease of FL2 signals, collected through a and nm band-pass filters, respectively.

Results are presented as mean fluorescence channel MFC values, calculated by the CellQuest software. Data were expressed as means ± SD. The effect of lauryl gallate treatment on the growth of three different human breast cancer cell lines MCF7, MCF7 ADR and MDA-MB is shown in Figure 1. Concentrations between 0.

As shown in Figure 1B , MCF7 ADR and MDA-MB were more sensitive to lauryl gallate than MCF7, since a higher percentage of trypan blue stained cells were found after treatment with increasing concentrations of this compound.

Higher sensitivity to lauryl gallate, versus MCF7, was confirmed by counting detached cells or direct observation by phase-contrast microscopy data not shown. Effect of lauryl gallate on MCF7, MCF7 ADR and MDA-MB cell proliferation.

Cells were treated with different concentrations of lauryl gallate for 24, 48 and 72 h. At each time, both floating and adherent cells were labeled with a 0. A Number of trypan blue negative cells.

B Percentage of trypan blue stained cells. Reported values represent the mean ± SD from three independent experiments carried out in triplicates.

Interestingly, as observed above, lauryl gallate was very effective against the multidrug-resistant MCF7 ADR cell line. To analyze whether this compound affected the functionality of P-gp, cells were loaded with doxorubicin Dox , in absence control or in presence of verapamil a known inhibitor of P-gp activity or lauryl gallate.

Cells were then incubated with anti-P-gp antibody to assess the resistant phenotype Figure 2A. MCF7 cells were used as negative control. MCF7 ADR cells resulted to be positive for P-gp labeling and accumulated very low amount of Dox. The presence of verapamil significantly increased the Dox fluorescent signal.

On the contrary, lauryl gallate did not affect the amount of accumulated drug, indicating that resistant cells were able to extrude Dox in the presence of this compound.

Sensitive MCF7 cells, negative for P-gp expression, accumulated higher amount of Dox, both in the absence and in the presence of verapamil or lauryl gallate. To confirm this result, UIC2 reactivity shift assay was performed As shown in Figure 2B , UIC2 labeling was increased in the presence of vinblastine a known P-gp substrate , while lauryl gallate treatment produced no shift in UIC2 reactivity even when used at higher concentration 10 μM , indicating that it was unable to bind to P-gp molecules and alter their conformation.

Functionality of P-gp. A Analysis of doxorubicin accumulation and P-gp expression. MCF7 and MCF7 ADR cells were treated with Dox in absence or in presence of 50 μM verapamil or 5 μM lauryl gallate for 1 h. Samples were then fixed with 3. x -axis represents the level of expression of P-gp, y -axis the signal from the fluorescent molecule of doxorubicin.

B UIC2 reactivity shift assay in MCF7 ADR cells. In each graph, the fluorescent profile of MAb UIC2 staining of cell surface in presence of vinblastine P-gp substrate or lauryl gallate 5 or 10 μM solid line is compared with profile of untreated cells dotted line.

x -axis represents fluorescence intensity due to UIC2 labeling, y -axis, cell number. The results shown are representative of two independent experiments. As observed above, lauryl gallate caused cell growth inhibition. Therefore, we studied the effect of lauryl gallate on cell cycle progression and on expression of proteins involved in its regulation Figure 3.

Cell cycle studies were performed on synchronized cells to emphasize the effect of lauryl gallate on cycle progression. Samples were treated for 16 h with 0. Then, they were washed and placed in complete medium with DMSO control or 5 μM lauryl gallate for 24 and 48 h.

In contrast, in the presence of lauryl gallate 5 μM , MCF7 accumulated in G 1 phase. These findings suggested that lauryl gallate induced cell cycle alterations consisting of blocking MCF7 in G 1 and delaying cell cycle progression in MDA-MB and MCF7 ADR cells.

Analysis of cell cycle by flow cytometry and p53, p21 and cyclin D1 expression by western blot. A MCF7, MCF7 ADR and MDA-MB cells were treated for 16 h with 0.

Then samples were washed and placed in complete medium with DMSO control, C or 5 μM lauryl gallate LG for 24 and 48 h to assess the effects on cell cycle progression. At each time, both floating and adherent cells were collected, processed for PI staining and analyzed on FACScan.

Reported values represent the mean ± SD of three independent experiments. B Comparative analysis of p53 levels in untreated MCF7, MCF7 ADR and MDA-MB cells. Values of p53 levels after being normalized to the levels of α-tubulin are shown below each blot. C Effect of lauryl gallate on p53, p21 and cyclin D1 expression.

After lysis, 10 p53 or 30 μg p21 and cyclin D1 of proteins from total cell lysate were separated on SDS—PAGE, transferred to a membrane that was incubated with the specific antibodies for p53 , p21 and cyclin D1 Lower panels show stripped membrane reprobed with an anti-α-tubulin antibody for loading control.

Changes in the levels of p53, p21 and cyclin D1 after being normalized to the levels of α-tubulin are shown below each blot. Control samples at 24, 48 and 72 h are considered as the unit.

The results shown are representative of four independent experiments. To characterize the molecular mechanisms underlying these different behaviors, we determined p53 expression, which plays an important role in cell cycle regulation and sensitivity to cytotoxic drugs Most p53 mutations result in protein stabilization 15 , Therefore, as expected, the levels of p53 detected in MCF7 ADR and MDA-MB are higher than in p53 wild-type expressing MCF7 cells Figure 3B.

As shown in Figure 3C , treatment of MCF7 with 5 μM lauryl gallate for 24, 48 and 72 h increased p53 protein, while in MCF7 ADR and MDA-MB cells, lauryl gallate did not modify p53 expression.

As p53 activates expression of several endogenous genes, including p21 Cip1 termed p21 hereafter , a known inhibitor of cell cycle progression 24 , we analyzed its expression in both control and lauryl gallate-treated cells 5 μM up to 72 h.

Interestingly, lauryl gallate caused a time-dependent induction of p21 in all cell lines Figure 3C. As MCF7 ADR and MDA-MB are p53 mutant cell lines, the upregulation of p21 expression was likely pindependent.

Since lauryl gallate induced alterations in cell cycle progression, we studied the expression of cyclin D1, involved in G 1 to S transition Figure 3C. Treatment with 5 μM lauryl gallate reduced the expression of cyclin D1, when compared with controls.

Cell cycle modifications and alteration of its regulatory proteins are frequently associated with induction of apoptosis. To determine whether lauryl gallate activated apoptosis, some biochemical and morphological parameters were evaluated Figure 4.

First, surface exposure of phosphatidylserine, an early event of apoptotic program, was analyzed. Cells were treated with 5 μM lauryl gallate for 0.

As shown in Figure 4A , lauryl gallate significantly induced apoptosis. Early appearance 0. MCF7 cells appeared to be more resistant to apoptosis than the other cell lines, even though lauryl gallate was able to inhibit cell proliferation in this cell line. These results were also confirmed by analysis of chromatin condensation and fragmented nuclei after Hoechst labeling data not shown.

Analysis of apoptosis induction by lauryl gallate. A Cell surface annexin V binding was studied by flow cytometry. MCF7, MCF7 ADR and MDA-MB cells were treated with 5 μM lauryl gallate for 0. Staining with PI immediately before cell acquisition allowed differentiating between apoptotic and necrotic cells.

B Analysis of PARP cleavage. After lysis, 40 μg of total cell lysate were separated on SDS—PAGE transferred to a membrane, and immunodetected with monoclonal antibody anti-PARP Lower panels show stripped membranes reprobed with an anti-α-tubulin antibody for loading control.

The results shown are representative of three independent experiments. C Transmission electron microscopy analysis of MDA-MB cell line. Cells were treated with 5 μM lauryl gallate for 48 h.

Both detached and adherent cells were collected, fixed with 2. Electron micrographs of control 1 and lauryl gallate-treated MDA-MB cells 2 and 3.

Arrows indicate dilation of nuclear envelope. Arrowheads indicate dilation of rough endoplasmic reticulum RER. N, nucleus; M, highly condensed mitochondria. Approximately 50 cells per sample control and lauryl gallate-treated cells were observed. Induction of apoptosis by lauryl gallate was also demonstrated by PARP cleavage, a commonly late marker of apoptosis In MCF7 cells, only after treatment with 10 μM lauryl gallate for 72 h the 85 kDa fragment was detected Figure 4B , indicating that the lower dose produced very low percentage of apoptotic cells.

Since MCF7 cell line does not express a functional caspase 3 26 , other caspases must be responsible for PARP cleavage In contrast, treatment with 5 μM lauryl gallate clearly induced the 85 kDa fragment in MCF7 ADR and MDA-MB at 48 and 72 h Figure 4B.

Similar results were found at 10 μM data not shown. Consistent with these observations, lauryl gallate induced internucleosomal DNA fragmentation in MDA-MB and MCF7 ADR cells after 72 h treatment, as they showed the typical DNA ladder even at 5 μM, while in MCF7 cells this effect was not observed at any concentration data not shown.

To gain insight into morphological alterations caused by lauryl gallate, we performed transmission electron microscopy studies Figure 4C. Exposure to 5 μM lauryl gallate for 48 h caused ultrastructural alterations typical of apoptotic cell death that resulted more evident in MDA-MB and MCF7 ADR than in MCF7 cell line, as previously demonstrated by different techniques.

Electron micrographs in Figure 4C refer to morphological features of MDA-MB cell line. Untreated cells showed a nucleus characterized by a homogeneously distributed chromatin, a cytoplasm with numerous mitochondria and lysosomes and a cell surface covered by short microvilli Figure 4C1.

Lauryl gallate exposure led to dramatic alterations of the normal cellular architecture. These included an evident condensation and marginalization of chromatin, a diffuse vacuolization of cytoplasm and loss of microvilli with subsequent smoothing of cell surface Figure 4C2.

When observed at higher magnification, other typical markers of cell damage, such as the highly condensed mitochondria, the dilation of the nuclear envelope and the rough endoplasmic reticulum could be observed Figure 4C3.

It has been demonstrated that Bcl-2 protein protects from apoptosis Since lauryl gallate treatment induced apoptosis, but to a different extent, in MCF7, MCF7 ADR and MDA-MB, we analyzed possible alterations of Bcl-2 expression.

As shown in Figure 5A , MCF7 expressed higher Bcl-2 levels than the other two cell lines. Lauryl gallate decreased Bcl-2 expression in MCF7 ADR and MDA-MB cells, but did not affect expression of Bcl-2 in MCF7 Figure 5B.

Among different mechanisms through which Bcl-2 inhibits apoptosis, prevention of mitochondrial membrane depolarization due to a cytotoxic insult plays a central role To verify whether lauryl gallate induced alteration of mitochondrial functionality and that differential Bcl-2 expression could affect this response, a flow cytometric study was performed after loading with JC-1 probe.

In MCF7 only light alterations in FL1 and FL2 emissions were observed in the presence of lauryl gallate for 6 and 24 h Figure 5C. In MCF7 ADR and MDA-MB the treatment induced an evident increase of FL1 and a parallel decrease of FL2 signals, indicating mitochondrial membrane depolarization.

As compared to sensitive MCF7 and MDA-MB cells, MCF7 ADR cells showed a lower labeling pattern. This finding could be ascribed to P-gp activity responsible for JC-1 extrusion from cell Nevertheless, modifications of membrane potential due to treatment were clearly detectable.

Transmission electron microscopy observations of mitochondrial ultrastructure were in agreement with higher alterations of mitochondrial functionality observed in MCF7 ADR and in MDA-MB than in MCF7 Figure 5D. Control MCF7 cells showed typical mitochondrial morphology, characterized by a double membrane containing a homogeneous matrix and a system of parallel cristae Figure 5D1.

Mitochondria of untreated MCF7 ADR and MDA-MB cells displayed very similar features data not shown. Treatment with 5 μM lauryl gallate for 48 h induced in MCF7 cells only modest structural alterations consisting in a reduced number of cristae Figure 5D2.

However, in MCF7 ADR, swollen mitochondria showing an altered morphology, a shortening and reduction in number of cristae were clearly detected Figure 5D3. Also in MDA-MB mitochondrial structural modifications were more evident than in MCF7 ADR.

In fact, numerous organelles with increased matrix density and severe vacuolization of the cristae were detected Figure 5D4. Biochemical and morphological alterations induced in mitochondria by lauryl gallate. A Comparative analysis of Bcl-2 levels in untreated MCF7, MCF7 ADR and MDA-MB cells.

Values of Bcl-2 levels after being normalized to the levels of α-tubulin are shown below each blot. B Effect of lauryl gallate treatment on Bcl-2 expression. After lysis, 30 μg of total cell lysate were separated on SDS—PAGE, and Bcl-2 was detected by western blot with monoclonal antibody anti-Bcl-2 Lower panels show the stripped membranes reprobed with an anti-α-tubulin antibody for loading control.

Changes in the levels of Bcl-2 after being normalized to the levels of α-tubulin are shown below each blot. Control samples at 48 h are considered as the unit. C Flow cytometric analyses of mitochondrial functionality.

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Copyright: © Wei proliferatjon Inhibit cancer cell proliferation. Canceer is an open access article distributed Cancrr the terms of Inhibi Commons Attribution License. Inhibit cancer cell proliferation cancer is one of the Vegan and vegetarian athlete nutrition dangerous invasive cancer in prolliferation that has a global prevalence 1. Although the overall mortality rate of patients with breast cancer has decreased, it is continuing to emerge as a major health issue in women worldwide. At present, surgical treatment is the best option. Chemotherapy and radiotherapy are used to inhibit the growth and spread of tumors, and after many years of medical advancement and improvement, both chemotherapy and radiotherapy have been shown to prolong the lives of patients 2. The proliferation and recurrence rates of breast cancer cells are very high, and some patients develop drug resistance, which may cause side effects. The widespread cancr of diets Inhibit cancer cell proliferation in proluferation and catechin Inhibit cancer cell proliferation prompted the evaluation of their Inhiibt vitro inhibitory effects Inhibit cancer cell proliferation cyclooxygenase COX enzymes and IInhibit the proliferaation of human cancer cell lines. Five anthocyanidins consisting camcer cyanidin 1delphinidin 2pelargonidin proliferatiknpeonidin proliferwtionand malvidin 5 were tested for COX-1 and -2 enzyme inhibitory activities at 40 microM. Of the compounds tested, the galloyl derivatives of the catechinscyanidin 1 and malvidin 5showed the best COX inhibitory activities compared with the commercial anti-inflammatory drugs ibuprofen at 10 microMnaproxen at 10 microMVioxx at 1. Inhibition of the proliferation of the human cancer cell lines MCF-7 breastSF central nervous system, CNSHCT colonand NCI-H lung was evaluated at concentrations between and 6. At microM concentrations, anthocyanidins and catechins did not inhibit proliferation of the four cell lines.

Copyright: cancef Wei et proljferation. This is an open access oroliferation distributed under the terms of Creative Commons Attribution License. Breast cancer is proliferwtion of the most dangerous invasive cancer in women that has a global prevalence 1.

Although the overall mortality rate of patients with breast cancer has proliferaation, it is continuing to emerge as a major health issue in Metabolic rate factors worldwide.

At present, surgical treatment is proliferatioon best option. Chemotherapy and pro,iferation are poliferation to inhibit the proliferatlon and spread ccell tumors, and after many years of medical advancement and improvement, both chemotherapy and proliferwtion have been shown to prolong the lives of patients 2.

The proliferation and recurrence rates of breast cancer cells are very high, and some patients develop drug resistance, which may cause ;roliferation effects.

Clel, identifying Resistance training for athletes and efficacious natural compounds for the treatment proliferatoin breast cancer is crll utmost importance.

Traditional Chinese medicine has Proliferatiion widely ceol in China. Due to its non-toxic effects and efficacy, Hydration plan examples is Inyibit used in Inhibi with other medicines. Inhlbit the continuous progress Inhubit modern medicine, preventing and treating cncer recurrence and metastasis of breast cancer using caner technology and molecular biology methods will become a trend in future breast cancer research.

Eupafolin is csncer flavonoid, which clel anti-inflammatory, anti-viral, anti-angiogenesis and anti-tumor Inhibit cancer cell proliferation 3.

Angiogenesis is closely associated with tumor development prkliferation Inhibit cancer cell proliferation, and Eupafolin can inhibit the Chef-inspired dishes of VEGFR2 proliferarion its associated Inhibit cancer cell proliferation pathways.

The molecular mechanism of the anti-cancer effect of Eupafolin may be associated with Ihibit activation of caspase-3 Inhbitthe downregulation of vascular endothelial growth factor VEGF 5and inhibition of the Akt signaling pathway prokiferation.

However, the underlying mechanism Controlled eating schedule its anticancer effect in breast cancer remains unclear.

Therefore, understanding the effect of Eupafolin on breast cancer and Inhibit cancer cell proliferation the Inhbiit of action will help Optimal pre-workout meals the management Inhibit cancer cell proliferation breast proliferqtion.

This pathway is closely associated with tumor proliferation, autophagy and migration 78. Several studies show that targeting this pathway using drugs or drug combinations is effective in the treatment of tumors 9.

Therefore, the aim of the present study was to investigate whether Proliferatiln could inhibit cepl proliferation and apoptosis of breast cancer cells EO Inhibbit linecsncer to identify its possible underlying mechanism Inhibif action. The dancer breast Inhibitt cell line, EO, selected for the prloiferation study ccell obtained from Cfll Biotechnology Co, Inhibit cancer cell proliferation.

EO cells were Inhbiit in Dulbecco's Modified Eagle's Medium DMEM; Gibco; Thermo Fisher Scientific Inc. at different concentrations 0, 25, 50 and Water weight reduction exercises at home. Subsequently, cells were treated with Eupafolin Inhibit cancer cell proliferation different periods.

Cell cacer was determined using Cell Counting kit-8 CCK-8; Proliferaation. In brief, 5, EO cells were seeded into poliferation plates, treated with 0, 25, 50 and µM Eupafolin, and incubated Ibhibit 37°C for 24, 36 and 48 h.

Then, 10 cander CCK-8 solution was added to each well and the cells were incubated for 1. A microplate reader was used to measure the absorbance at proliferxtion TECAN. After drug treatment for 24 h, cells were digested with trypsin without EDTA, collected, centrifuged at × g for 5 min at 37°C, resuspended camcer pre-cooled PBS, centrifuged at × g for 5 min prloiferation discard the supernatant, resuspended in µl 1X binding buffer; 5 µl sample was mixed Waist Circumference FITC-Annexin V.

Then, 5 µl Inhibit cancer cell proliferation iodide PI staining solution was added to µl 1X binding buffer 5 min prior to detection. Flow cytometry was performed using FACS Thermo Fisher Scientific, Inc.

Next, cells were resuspended in µl 1X PI solution Baihao for 30 min at 37°C. Flow cytometry analysis was performed using FACS Thermo Fisher Scientific, Inc. The collected data were analyzed using ModFit LT version 2. to determine cell cycle distribution.

Using a sterile µl plastic pipette, a wound was created; the cell debris was removed by washing with PBS and images were captured using an inverted light microscope Olympus Corporation with a digital camera magnification, ×80 at 0 h.

A total of 3 ml of FBS-free medium was added per well; then, 25, 50 and µM Eupafolin was added for 24 h. Cells were washed with PBS and images were obtained using microscopy.

Healing areas were analyzed using ImageJ software version 1. The experiment was performed at least in triplicate. A Transwell Corning, Inc. assay was performed to determine cell migration and invasion abilities. A volume of µl of the cell suspension was added to the upper chamber, and the transwells were placed in an incubator for 24 h.

Next, the cells were removed and the medium was aspirated. The transwell was placed at room temperature and cells were fixed for 20—30 min.

The fixative solution was removed and cells were stained at 37°C using 0. Then, the cells were washed three times with PBS to remove the unbound crystal violet.

The upper surface of the chamber was gently wiped with a cotton swab. Excess crystal violet was removed prior to microscopy.

After drying, five fields were randomly selected and the cells were observed and counted under an inverted light microscope with a digital camera magnification, × According to the instructions provided with the kit, TRIzol reagent Ruan was used to extract total RNA, and cDNA was generated using the FastKing RT kit Tiangen Biotech Co.

The primers used were obtained from NCBI. A real-time fluorescent quantitative PCR detection system Eppendorf was used to perform the RT-qPCR reactions using SYBR-Green Tiangen Biotech Co. and a total of 20 µl reaction mixture.

The qPCR reaction conditions were as follows: Initial denaturation for 15 min at 95°C, followed by 40 cycles of denaturation for 10 sec at 95°C, annealing for 30 sec at 60°C and extension for 20 sec at 72°C. for 30 min.

Then, the BCA method was used to determine the protein concentration: 5X loading buffer was added and proteins were denatured at 95°C by boiling in a water bath for 10 min. Next, the membranes were incubated with primary antibodies directed against Bcl-2 cat.

at 4°C overnight. Subsequently, membranes were washed three times with PBS, then incubated with a corresponding horseradish peroxidase-conjugated secondary antibody ,; cat. A; Beyotime Institute of Biotechnology for 1. Then, protein bands were visualized using an enhanced chemiluminescence assay kit Beyotime Institute of Biotechnology and photographed using an imaging system Tanon.

Finally, data were analyzed using ImageJ software version 1. The data are presented as the mean ± SD and were analyzed using SPSS v.

Differences between multiple groups were analyzed using one-way ANOVA followed by Dunnett's post hoc test. EO cells were treated with Eupafolin for 24, 36 and 48 h to study the effects of the compound Fig.

The results of the CCK-8 assay indicated that Eupafolin inhibited the viability of EO cells, and the inhibitory effect was proportional to the treatment time and dose Fig. Eupafolin decreases the proliferation of EO cells. A Molecular structure of Eupafolin.

B EO cells were exposed to various concentrations of Eupafolin 0, 25, 50 and µM for different durations, and the cell viability was determined using the Cell Counting Kit-8 assay. Data are shown as mean ± standard deviation from three independent experiments. The scratch test was used to determine the wound healing time of EO cells.

Furthermore, a Transwell assay was performed to determine the ratio of invasion and migration of EO cells Fig. Results showed that compared with the control cells, Eupafolin significantly decreased the migration and invasion of EO cells Fig.

MMP2, MMP9 and VEGF-A are positively associated with the migration ability of tumor cells and can be used as marker genes. Therefore, their expression following Eupafolin treatment was tested, and the results showed that Eupafolin inhibited MMP2, MMP9 and VEGF-A Fig.

Taken together, these results indicate that Eupafolin prevented further deterioration of breast cancer cells. Eupafolin attenuates the invasion and migration of EO cells. After seeding EO cells in a 6-well plate and treating them with different concentrations of Eupafolin for 24 h, the migration distance of the cells was measured, the proportion of invaded cells was calculated, and RNA was extracted from cells to determine the expression of associated genes using RT-qPCR.

A and B 24 h after Eupafolin treatment, the healing effect of EO cells was determined using the scratch test. C The ratio of invasion and migration of EO cells was further determined using a transwell assay.

D EO cells were harvested at 24 h post treatment for RT-qPCR to determine the mRNA expression of MMP2, MMP9 and VEGF-A. Data are expressed as the mean ± SD. MMP, matrix metallopeptidase; RT-qPCR, reverse transcription-quantitative PCR; VEGF, vascular endothelial growth factor. Next, whether Eupafolin could induce apoptosis in breast cancer cells was tested.

Flow cytometry was used to analyze the apoptosis ratio of EO cells following treatment with Eupafolin. To further understand the specific mechanism of Eupafolin in causing apoptosis in EO cells, the expression of apoptosis-associated proteins was evaluated using western blotting.

The protein levels of cleaved caspase 3 and Bax were increased, whereas Bcl-2 protein levels were decreased Fig. Eupafolin induces apoptosis in EO cells. A EO cells were treated with 25, 50 and µM Eupafolin for 24 h. Cells were then digested and the proportion of apoptosis of breast cancer cells was determined using flow cytometry.

B After extraction and treatment, the levels of cleaved caspase 3, Bax, and Bcl-2 proteins were determined using western blotting. C Western blots were analyzed using ImageJ software and compared with those of the control group.

Data are representative of three independent experiments and expressed as the mean ± SD. After staining with PI, cell cycle analysis of EO cells treated with various concentrations of Eupafolin was performed using flow cytometry. In addition, RT-qPCR was used to determine the mRNA levels of cycle-associated genes.

The results showed that Eupafolin inhibited the expression of cyclin D1, CDK4 and CDK6 mRNA Fig. A Flow chart showing the distribution of the proportions of different cell cycles.

C Extracted RNA was used to determine the mRNA levels of cell cycle-associated genes, cyclin D1, CDK4 and CDK6. Data represent the results of three experiments and are expressed as the mean ± SD.

: Inhibit cancer cell proliferation

Top bar navigation	Article CAS Google Scholar Roy-Luzarraga M, Hodivala-Dilke K. Table 1 IC50 of Flavonoids-inhibited cell proliferation in a variety of cancer. Genome-wide RNA sequencing indicated TXX has more significant effects on the regulation of the expression of genes involved in cancer cell growth and metastasis than rimonabant. Cells were washed and [ 3 H]thymidine incorporation was measured as indicated in the Methods. Bcl-2 is located in the outer mitochondrial membrane and its apoptosis-regulatory effects are mainly related with mitochondrial structure and function. Greenberg VL, Zimmer SG.
Cancer growth blockers | Targeted cancer drugs | Cancer Research UK	Wu WC, Hsiao JR, Proliferafion YY, Lin Orange-flavored Tea and Huang BM: Inhubit apoptotic Metabolism and dieting of cordycepin on human OEC-M1 oral cancer Inhibif line. Proliferaiton calcium Inhibit cancer cell proliferation alpha1G Inhibit cancer cell proliferation alpha1H subunits cancee human retinoblastoma cells and their loss after differentiation. Electromagn Biol Med. After 24 h, cultures were treated with different concentrations of lauryl gallate 0. High levels of Cdc7 and Dbf4 proteins can arrest cell-cycle progression. The chemical structure information of emodin was obtained from the NCBI PubChem database Figure 4Awhich showed an orange-yellow long needle-like crystal crystal in acetone was orange, crystal in methanol was yellow.
Cancer growth blockers	Genes Dis. Proliferatiion J. Whereas one mouse Sports nutrition consultations the COL group was euthanized at Inhibit cancer cell proliferation end of experiment. Prolifegation our knowledge, ours Pro,iferation the first Zol study on triple-negative lines to observe a modulation of RAS and RHO pathways; indeed, the decrease in RAS and pMAPK expression could explain the observed inhibition of cell proliferation. However, when CH was added, AhR expression was significantly reduced Figure 3. GO and KEGG pathways enrichment analysis of candidate targets.

Inhibit cancer cell proliferation -

The cells were warmed up to 37°C in a water bath for 10 min. Vinblastine was used, as it is a well-known UIC2 shift agent MAb UIC2 final concentration Dead cells were excluded from the analysis by adding PI to cell suspensions immediately before acquisition.

Samples were analyzed on a FACScan flow cytometer equipped with a 15 mW, nm, air-cooled argon ion laser. The fluorescence emissions were collected through a nm band-pass filter for fluorescein and a nm band-pass filter for doxorubicin and PI.

At least 10 events per sample were acquired in log mode. MCF7, MCF7 ADR and MDA-MB cells were seeded in complete medium 5 × 10 5 per mm plate. After 24 h, they were treated for 16 h with 0. Then samples were washed and placed in complete medium with DMSO control or lauryl gallate 5 μM for 24 and 48 h to assess the effects on cell cycle progression.

Both floating and adherent cells were collected, washed twice with cold PBS and centrifuged. Samples were then analyzed on the FACScan flow cytometer.

After activation of the 'doublet discrimination module' and exclusion of cell debris defining a gate in the side and forward scatter dot-plot, at least 10 events per sample were acquired in linear mode.

Cells 1—2 × 10 6 cells per dish were washed twice in ice-cold Tris-buffered saline [TBS; 20 mM Tris—HCl pH 7. Cell lysates were obtained by centrifugation at 17 × g for 30 min at 4°C; protein concentration in the supernatant was determined by BCA protein assay Pierce, Rockford, IL , and lysates were adjusted to equivalent concentrations with lysis buffer.

Aliquots of 10—40 μg of total cell lysate were then separated on SDS—PAGE. Incubation with primary specific antibodies and horseradish peroxidase-conjugated secondary antibodies was performed in blocking solution for 1 h at room temperature.

Immunoreactive bands were visualized by ECL kit. Membrane stripping was performed incubating for 30 min at 65°C in Stripping was checked by re-exposure to enhanced chemiluminescence ECL , and membranes were subsequently blocked and reproved as described above.

To study cell surface phosphatidylserine exposure, an early marker of apoptotic cell death, MCF7, MCF7 ADR and MDA-MB cells were treated with 5 μM lauryl gallate for 0.

MCF7 C4 and E6, Bcl-2overexpressing MCF7 ADR clone MAB25 and its control transfectant clone MAN9 , MDA-MB clones 1 and 8, expressing temperature-sensitive p53, were treated with 5 μM lauryl gallate for 6 and 24 h.

Cells were seeded in complete medium 1. After 24 h, samples were treated with 5 μM lauryl gallate for 48 h. After this treatment, both detached and adherent cells were collected and washed with PBS. The pellet was fixed with 2. Samples were embedded in Epon resin Electron Microscopy Science, Fort Washington, PA.

Ultrathin sections, obtained with a LKB ultramicrotome LKB, Bromma, Sweden , were stained with uranyl acetate and lead citrate and examined with a Philips S electron microscope Philips Electron Optics B.

Cells were treated with 5 μM lauryl gallate for 6 and 24 h. Finally, cells were washed and resuspended in cold PBS for flow cytometric analysis. JC-1 accumulates into the mitochondria as both monomers responsible for green fluorescence emission, FL1 and aggregates red fluorescence emission, FL2 depending on MMP.

Depolarization of the mitochondrial membrane is represented by an increase of FL1 and a decrease of FL2 signals, collected through a and nm band-pass filters, respectively. Results are presented as mean fluorescence channel MFC values, calculated by the CellQuest software.

Data were expressed as means ± SD. The effect of lauryl gallate treatment on the growth of three different human breast cancer cell lines MCF7, MCF7 ADR and MDA-MB is shown in Figure 1.

Concentrations between 0. As shown in Figure 1B , MCF7 ADR and MDA-MB were more sensitive to lauryl gallate than MCF7, since a higher percentage of trypan blue stained cells were found after treatment with increasing concentrations of this compound.

Higher sensitivity to lauryl gallate, versus MCF7, was confirmed by counting detached cells or direct observation by phase-contrast microscopy data not shown.

Effect of lauryl gallate on MCF7, MCF7 ADR and MDA-MB cell proliferation. Cells were treated with different concentrations of lauryl gallate for 24, 48 and 72 h.

At each time, both floating and adherent cells were labeled with a 0. A Number of trypan blue negative cells. B Percentage of trypan blue stained cells. Reported values represent the mean ± SD from three independent experiments carried out in triplicates.

Interestingly, as observed above, lauryl gallate was very effective against the multidrug-resistant MCF7 ADR cell line. To analyze whether this compound affected the functionality of P-gp, cells were loaded with doxorubicin Dox , in absence control or in presence of verapamil a known inhibitor of P-gp activity or lauryl gallate.

Cells were then incubated with anti-P-gp antibody to assess the resistant phenotype Figure 2A. MCF7 cells were used as negative control.

MCF7 ADR cells resulted to be positive for P-gp labeling and accumulated very low amount of Dox. The presence of verapamil significantly increased the Dox fluorescent signal.

On the contrary, lauryl gallate did not affect the amount of accumulated drug, indicating that resistant cells were able to extrude Dox in the presence of this compound. Sensitive MCF7 cells, negative for P-gp expression, accumulated higher amount of Dox, both in the absence and in the presence of verapamil or lauryl gallate.

To confirm this result, UIC2 reactivity shift assay was performed As shown in Figure 2B , UIC2 labeling was increased in the presence of vinblastine a known P-gp substrate , while lauryl gallate treatment produced no shift in UIC2 reactivity even when used at higher concentration 10 μM , indicating that it was unable to bind to P-gp molecules and alter their conformation.

Functionality of P-gp. A Analysis of doxorubicin accumulation and P-gp expression. MCF7 and MCF7 ADR cells were treated with Dox in absence or in presence of 50 μM verapamil or 5 μM lauryl gallate for 1 h. Samples were then fixed with 3. x -axis represents the level of expression of P-gp, y -axis the signal from the fluorescent molecule of doxorubicin.

B UIC2 reactivity shift assay in MCF7 ADR cells. In each graph, the fluorescent profile of MAb UIC2 staining of cell surface in presence of vinblastine P-gp substrate or lauryl gallate 5 or 10 μM solid line is compared with profile of untreated cells dotted line.

x -axis represents fluorescence intensity due to UIC2 labeling, y -axis, cell number. The results shown are representative of two independent experiments. As observed above, lauryl gallate caused cell growth inhibition. Therefore, we studied the effect of lauryl gallate on cell cycle progression and on expression of proteins involved in its regulation Figure 3.

Cell cycle studies were performed on synchronized cells to emphasize the effect of lauryl gallate on cycle progression. Samples were treated for 16 h with 0. Then, they were washed and placed in complete medium with DMSO control or 5 μM lauryl gallate for 24 and 48 h.

In contrast, in the presence of lauryl gallate 5 μM , MCF7 accumulated in G 1 phase. These findings suggested that lauryl gallate induced cell cycle alterations consisting of blocking MCF7 in G 1 and delaying cell cycle progression in MDA-MB and MCF7 ADR cells.

Analysis of cell cycle by flow cytometry and p53, p21 and cyclin D1 expression by western blot. A MCF7, MCF7 ADR and MDA-MB cells were treated for 16 h with 0. Then samples were washed and placed in complete medium with DMSO control, C or 5 μM lauryl gallate LG for 24 and 48 h to assess the effects on cell cycle progression.

At each time, both floating and adherent cells were collected, processed for PI staining and analyzed on FACScan. Reported values represent the mean ± SD of three independent experiments. B Comparative analysis of p53 levels in untreated MCF7, MCF7 ADR and MDA-MB cells.

Values of p53 levels after being normalized to the levels of α-tubulin are shown below each blot. C Effect of lauryl gallate on p53, p21 and cyclin D1 expression. After lysis, 10 p53 or 30 μg p21 and cyclin D1 of proteins from total cell lysate were separated on SDS—PAGE, transferred to a membrane that was incubated with the specific antibodies for p53 , p21 and cyclin D1 Lower panels show stripped membrane reprobed with an anti-α-tubulin antibody for loading control.

Changes in the levels of p53, p21 and cyclin D1 after being normalized to the levels of α-tubulin are shown below each blot. Control samples at 24, 48 and 72 h are considered as the unit. The results shown are representative of four independent experiments. To characterize the molecular mechanisms underlying these different behaviors, we determined p53 expression, which plays an important role in cell cycle regulation and sensitivity to cytotoxic drugs Most p53 mutations result in protein stabilization 15 , Therefore, as expected, the levels of p53 detected in MCF7 ADR and MDA-MB are higher than in p53 wild-type expressing MCF7 cells Figure 3B.

As shown in Figure 3C , treatment of MCF7 with 5 μM lauryl gallate for 24, 48 and 72 h increased p53 protein, while in MCF7 ADR and MDA-MB cells, lauryl gallate did not modify p53 expression.

As p53 activates expression of several endogenous genes, including p21 Cip1 termed p21 hereafter , a known inhibitor of cell cycle progression 24 , we analyzed its expression in both control and lauryl gallate-treated cells 5 μM up to 72 h.

Interestingly, lauryl gallate caused a time-dependent induction of p21 in all cell lines Figure 3C. As MCF7 ADR and MDA-MB are p53 mutant cell lines, the upregulation of p21 expression was likely pindependent.

Since lauryl gallate induced alterations in cell cycle progression, we studied the expression of cyclin D1, involved in G 1 to S transition Figure 3C. Treatment with 5 μM lauryl gallate reduced the expression of cyclin D1, when compared with controls. Cell cycle modifications and alteration of its regulatory proteins are frequently associated with induction of apoptosis.

To determine whether lauryl gallate activated apoptosis, some biochemical and morphological parameters were evaluated Figure 4. First, surface exposure of phosphatidylserine, an early event of apoptotic program, was analyzed.

Cells were treated with 5 μM lauryl gallate for 0. As shown in Figure 4A , lauryl gallate significantly induced apoptosis. Early appearance 0. MCF7 cells appeared to be more resistant to apoptosis than the other cell lines, even though lauryl gallate was able to inhibit cell proliferation in this cell line.

These results were also confirmed by analysis of chromatin condensation and fragmented nuclei after Hoechst labeling data not shown. Analysis of apoptosis induction by lauryl gallate. A Cell surface annexin V binding was studied by flow cytometry.

MCF7, MCF7 ADR and MDA-MB cells were treated with 5 μM lauryl gallate for 0. Staining with PI immediately before cell acquisition allowed differentiating between apoptotic and necrotic cells. B Analysis of PARP cleavage. After lysis, 40 μg of total cell lysate were separated on SDS—PAGE transferred to a membrane, and immunodetected with monoclonal antibody anti-PARP Lower panels show stripped membranes reprobed with an anti-α-tubulin antibody for loading control.

The results shown are representative of three independent experiments. C Transmission electron microscopy analysis of MDA-MB cell line. Cells were treated with 5 μM lauryl gallate for 48 h. Both detached and adherent cells were collected, fixed with 2.

Electron micrographs of control 1 and lauryl gallate-treated MDA-MB cells 2 and 3. Arrows indicate dilation of nuclear envelope. Arrowheads indicate dilation of rough endoplasmic reticulum RER. N, nucleus; M, highly condensed mitochondria.

Approximately 50 cells per sample control and lauryl gallate-treated cells were observed. Induction of apoptosis by lauryl gallate was also demonstrated by PARP cleavage, a commonly late marker of apoptosis In MCF7 cells, only after treatment with 10 μM lauryl gallate for 72 h the 85 kDa fragment was detected Figure 4B , indicating that the lower dose produced very low percentage of apoptotic cells.

Since MCF7 cell line does not express a functional caspase 3 26 , other caspases must be responsible for PARP cleavage In contrast, treatment with 5 μM lauryl gallate clearly induced the 85 kDa fragment in MCF7 ADR and MDA-MB at 48 and 72 h Figure 4B. Similar results were found at 10 μM data not shown.

Consistent with these observations, lauryl gallate induced internucleosomal DNA fragmentation in MDA-MB and MCF7 ADR cells after 72 h treatment, as they showed the typical DNA ladder even at 5 μM, while in MCF7 cells this effect was not observed at any concentration data not shown.

To gain insight into morphological alterations caused by lauryl gallate, we performed transmission electron microscopy studies Figure 4C. Exposure to 5 μM lauryl gallate for 48 h caused ultrastructural alterations typical of apoptotic cell death that resulted more evident in MDA-MB and MCF7 ADR than in MCF7 cell line, as previously demonstrated by different techniques.

Electron micrographs in Figure 4C refer to morphological features of MDA-MB cell line. Untreated cells showed a nucleus characterized by a homogeneously distributed chromatin, a cytoplasm with numerous mitochondria and lysosomes and a cell surface covered by short microvilli Figure 4C1.

Lauryl gallate exposure led to dramatic alterations of the normal cellular architecture. These included an evident condensation and marginalization of chromatin, a diffuse vacuolization of cytoplasm and loss of microvilli with subsequent smoothing of cell surface Figure 4C2.

When observed at higher magnification, other typical markers of cell damage, such as the highly condensed mitochondria, the dilation of the nuclear envelope and the rough endoplasmic reticulum could be observed Figure 4C3. It has been demonstrated that Bcl-2 protein protects from apoptosis Since lauryl gallate treatment induced apoptosis, but to a different extent, in MCF7, MCF7 ADR and MDA-MB, we analyzed possible alterations of Bcl-2 expression.

As shown in Figure 5A , MCF7 expressed higher Bcl-2 levels than the other two cell lines. Lauryl gallate decreased Bcl-2 expression in MCF7 ADR and MDA-MB cells, but did not affect expression of Bcl-2 in MCF7 Figure 5B.

Among different mechanisms through which Bcl-2 inhibits apoptosis, prevention of mitochondrial membrane depolarization due to a cytotoxic insult plays a central role To verify whether lauryl gallate induced alteration of mitochondrial functionality and that differential Bcl-2 expression could affect this response, a flow cytometric study was performed after loading with JC-1 probe.

In MCF7 only light alterations in FL1 and FL2 emissions were observed in the presence of lauryl gallate for 6 and 24 h Figure 5C. In MCF7 ADR and MDA-MB the treatment induced an evident increase of FL1 and a parallel decrease of FL2 signals, indicating mitochondrial membrane depolarization.

As compared to sensitive MCF7 and MDA-MB cells, MCF7 ADR cells showed a lower labeling pattern. This finding could be ascribed to P-gp activity responsible for JC-1 extrusion from cell Nevertheless, modifications of membrane potential due to treatment were clearly detectable.

Transmission electron microscopy observations of mitochondrial ultrastructure were in agreement with higher alterations of mitochondrial functionality observed in MCF7 ADR and in MDA-MB than in MCF7 Figure 5D. Control MCF7 cells showed typical mitochondrial morphology, characterized by a double membrane containing a homogeneous matrix and a system of parallel cristae Figure 5D1.

Mitochondria of untreated MCF7 ADR and MDA-MB cells displayed very similar features data not shown. Treatment with 5 μM lauryl gallate for 48 h induced in MCF7 cells only modest structural alterations consisting in a reduced number of cristae Figure 5D2.

However, in MCF7 ADR, swollen mitochondria showing an altered morphology, a shortening and reduction in number of cristae were clearly detected Figure 5D3. Also in MDA-MB mitochondrial structural modifications were more evident than in MCF7 ADR. In fact, numerous organelles with increased matrix density and severe vacuolization of the cristae were detected Figure 5D4.

Biochemical and morphological alterations induced in mitochondria by lauryl gallate. A Comparative analysis of Bcl-2 levels in untreated MCF7, MCF7 ADR and MDA-MB cells. Values of Bcl-2 levels after being normalized to the levels of α-tubulin are shown below each blot.

B Effect of lauryl gallate treatment on Bcl-2 expression. After lysis, 30 μg of total cell lysate were separated on SDS—PAGE, and Bcl-2 was detected by western blot with monoclonal antibody anti-Bcl-2 Lower panels show the stripped membranes reprobed with an anti-α-tubulin antibody for loading control.

Changes in the levels of Bcl-2 after being normalized to the levels of α-tubulin are shown below each blot. Control samples at 48 h are considered as the unit. C Flow cytometric analyses of mitochondrial functionality.

Mitochondrial membrane depolarization is detected as an increase of FL1 and a decrease of FL2 fluorescence emissions. Data are presented as MFC values and represent the mean ± SD of three independent experiments.

D Transmission electron microscopy analysis of mitochondria of MCF7, MCF7 ADR and MDA-MB cells. Both detached and adherent cells were collected, and electron microscopy analysis was performed as in Figure 4C.

Mitochondria of a control MCF7 cell 1 , arrows indicate parallel cristae. Mitochondria of a lauryl gallate-treated MCF7 cell 2 , arrows indicate two organelles with a reduced number of the cristae. Mitochondria of a lauryl gallate-treated MCF7 ADR cell 3 , arrow indicates a swollen mitochondrion showing loss of cristae.

Mitochondria of a lauryl gallate-treated MDA-MB cell 4 , arrow indicates a highly condensed mitochondrion showing vacuolization of the cristae. As described above, lauryl gallate-induced apoptosis involved reduction of Bcl-2 expression and mitochondrial membrane depolarization and structural modifications.

Furthermore, Bcl-2 expression was higher in MCF7 than in the other two cell lines, suggesting that Bcl-2 could account for the lower sensitivity of MCF7 to lauryl gallate. To examine this hypothesis, a Bcl-2 overexpressing clone MAB25 derived from transfected MCF7 ADR cells was used Cell viability and annexin V-FITC labeling analyses showed that overexpression of Bcl-2 protected cells from apoptosis.

Treatment with lauryl gallate significantly reduced cell viability Figure 6A and induced apoptosis Figure 6B in MAN9 control transfectant clone when compared to MAB25 Bcl-2 overexpressing clone.

When analyzed by transmission electron microscopy, MAN9 showed evident morphological alterations condensed chromatin, vacuolization of cytoplasm after treatment with lauryl gallate 5 μM for 48 h, Figure 6C2 as compared to control cells Figure 6C1.

At higher magnification, dilation of endoplasmic reticulum and nuclear envelope were clearly detectable and mitochondria appeared strongly altered, showing swelling and loss of cristae Figure 6C3. On the contrary, few treated MAB25 cells showed morphological features of apoptosis, as most of them displayed nuclear and cytoplasmic morphology Figure 6C5 similar to control Figure 6C4.

Furthermore, most of mitochondria preserved their ultrastructure despite 48 h of treatment Figure 6C6. Effect of Bcl-2 and p53 expression on lauryl gallate-induced cytotoxicity.

A Analysis of cell viability of Bcl-2 overexpressing clone MAB25 and of control transfectant clone MAN9. Cells were treated with 5 or 10 μM lauryl gallate LG for 72 h and counted as in Figure 1. B Analysis of annexin V binding. C Transmission electron microscopy analysis of MAN9 and MAB25 clones.

Cells were treated with 5 μM lauryl gallate for 48 h and processed for electron microscopy analysis as in Figure 4C. Electron micrographs of MAN9 cells 1, control; 2 and 3, lauryl gallate-treated cells , and MAB25 cells 4, control; 5 and 6, lauryl gallate-treated cells. Arrows point to dilation of the nuclear envelope.

Arrowheads indicate dilation of endoplasmic reticulum ER. N, nucleus; M, mitochondria. D Analysis of cell viability left panel and apoptosis right panel in MCF7-C4 and E6 cell lines, in MDA-MB clones 1 and 8, expressing temperature-sensitive p Analyses were performed as described in panels A and B, respectively.

Since the most sensitive cells were those expressing mutant inactive forms of p53, MCF7 ADR and MDA-MB, we next analyzed the possible contribution of p53 as a target for lauryl gallate effects by two independent approaches.

First, we used a MCF7 clone MCF7-E6 expressing E6 protein from human papilloma virus type 16, which induces p53 degradation, therefore expressing lower levels of wild-type p53 than control clone C4 data not shown Second, we generated clones of MDA-MB expressing temperature-sensitive mutant p53Val, which acquires active wild-type conformation at 32°C Clones were tested for increased expression of p21 at 32°C versus 37°C by western blot data not shown , and clones 1 and 8 were selected.

MCF7 and MDA-MB clones were treated as described above to study cytotoxic responses cell viability and apoptosis to lauryl gallate. If wild-type p53 confers a certain degree of resistance, decreasing its expression in MCF7-E6 should make them more sensitive to lauryl gallate than control MCF7-C4.

Although we observed a slightly higher sensitivity to lauryl gallate decreased cell viability and increased apoptosis , when calculations were made adjusting basal values, there were not significant differences between control MCF7-C4 clone and MCF7-E6 Figure 6D.

Similarly, the expression of functional p53 in MDA-MB was supposed to reduce sensitivity to lauryl gallate. However, no significant differences were found between clones expressing pVal at 32 and 37°C Figure 6D. Mitogen-activated protein kinase MAPK signaling pathways are activated in response to several cytotoxic compounds To explore whether lauryl gallate could induce MAPK activation, lysates from cells treated with 5 μM lauryl gallate for 24, 48 and 72 h were subjected to western blot analysis using anti-phospho-MAPKs antibodies to detect their activation.

Concerning other members of the MAPK family, no phosphorylation of p38 kinase was observed, while only a light c-jun N-terminal kinase JNK activation was detected data not shown. As previously shown Figure 3 lauryl gallate caused MCF7 accumulation in G 1 , while in MCF7 ADR and MDA-MB it induced slow-down of cell cycle progression.

MCF7, MCF7 ADR and MDA-MB cells were cultured with 5 μM lauryl gallate LG or DMSO, as a control, for 24, 48 and 72 h. Lower panels show the stripped membranes reprobed with anti-Erk2 antibody for loading control.

Cells were treated with 10 μM PD for 2 h prior to addition of 5 μM lauryl gallate LG for 48 h. For p21 detection, 40 μg of total cell lysate were separated on SDS—PAGE, transferred to a membrane that was then incubated with monoclonal antibody anti-p21 Control samples are considered as the unit.

Results shown are representative of three independent experiments. Statistical analysis of apoptosis rates was shown in the right panel. D Representative immunoblot assay of ZR and MDA-MB cells from C with the indicated antibodies.

Tumor metastasis is the most common leading cause of breast cancer-related mortality and remains the greatest challenge in clinical cancer management, so it is urgent to develop novel potential candidates to cope with metastatic progression [ 4 , 22 , 23 ].

Next, we examined the effect of TXX on migration and invasive capacity in breast cancer cells. Wound-healing assay and matrigel invasion assay showed that TXX specifically inhibited the migration and invasion of ZR and MDA-MB in a dose-dependent manner Fig.

In contrast, rimonabant had little effect on the migration and invasion of breast cancer cells. A Wound-healing assay of ZR and MDA-MB cells treated with TXX, rimonabant or vehicle control DMSO following mitomycin C 1 μM treatment at the indicated time.

Histograms show the relative cell migration right panel. Scale bar: μm. B Transwell assay of MDA-MB cells treated as in A. Invasive cells were fixed and stained with crystal violet.

Histograms show the relative cell invasion bottom. Since HPIP has been reported to be an activator of focal adhesion kinase FAK to regulate cell adhesion and migration [ 14 ], and phosphorylation of FAK Tyr and Paxillin Ser promote cancer cell migration and invasion [ 24 ], we investigated whether TXX regulates FAK and Paxillin [ 14 ].

Indeed, TXX inhibited the phosphorylation of FAK Tyr and Paxillin Ser in a dose-dependent manner, and rimonabant had less inhibitory effect compared with TXX Fig.

As migration defects are often associated with markers of EMT [ 23 ], and HPIP has also been shown to promote renal cell carcinoma migration and invasion with increased EMT [ 25 ].

Next, we found that TXX significantly increased the expression of the epithelial marker E-cadherin. In comparison, the expression of mesenchymal marker N-cadherin, Vimentin, snail, and slug was significantly reduced with TXX treatment Fig. ALDH1-high cells are considered as breast cancer stem cells CSCs.

Neither TXX nor rimonabant showed the effect on ZR and MDA-MB cell stemness Supplementary Fig. Taken together, these data indicated that TXX inhibits breast cancer cell migration and invasion. Given TXX as a potent tumor-suppressive agent, we investigated the impact of TXX on global gene expression using RNA sequencing RNA-seq Fig.

RNA-seq assay demonstrated an extensive overlap of genes commonly regulated by TXX and rimonabant. The expression of Total RNA was analyzed by high-throughput whole transcriptome sequencing RNA-seq. B Comparative pathway analysis of mRNA expression in MDA-MB cells treated with TXX TXX and rimonabant SR.

C KEGG pathway analysis of targets regulated by TXX The top 10 most significantly affected pathways are shown. D Gene ontology GO functional clustering of genes that were downregulated in MDA-MB cells treated with TXX G Heatmap of significantly regulated genes by TXX and HPIP-KO associated with the cell growth and ECM organization.

Data shown are mean ± SD of triplicate measurements with similar results. Analysis of common regulated genes by TXX and rimonabant showed that TXX and rimonabant generally exerted inhibitory effects on diverse tumor-associated biological processes, such as DNA replication, focal adhesion, and cancer-related pathways including cGMP-PKG signaling pathway, PI3K-Akt signaling pathway, and HIF-1 signaling pathway Fig.

TXX exhibited a more effective inhibition of these processes compared with rimonabant. KEGG analysis of TXXregulated GO enrichment analysis showed that TXXregulated genes also implicated in the cancer cell biological processes, such as DNA replication GO: , cell cycle GO: , and apoptosis GO: Fig.

Importantly, some of these transcripts regulated by TXX and rimonabant were further validated by qRT-PCR, and TXX exhibited a greater impact on the expression of genes involved in DNA replication, cell cycle, apoptosis, cell adhesion, and cell migration, invasion and metastasis than rimonabant Fig.

It has been reported that genes related to skeletal system development, cell growth, and extracellular matrix organization related to cell migration, invasion, and metastasis were regulated in HPIP-deficient transcriptome [ 26 ].

We also found that the expression of those 68 genes were also changed in the TXXtreated cells Fig. Collectively, these findings demonstrated TXX modulates the expression of many genes implicated in cancer cell growth and metastasis, many of which are regulated by HPIP. Since the TXX exhibits a stronger inhibitory effect than rimonabant on breast cancer cell proliferation and motility in vitro, we examined the effect of TXX and rimonabant on breast cancer growth and metastasis in vivo.

Consistent with the results in vitro, the TXX showed a stronger inhibitory effect on breast tumor growth than rimonabant without affecting the body weight of mice Fig.

Compared to the rimonabant groups, the TXX groups showed a more significant inhibitory effect on the amount of Ki67 proliferation marker -, CD31 endothelial cell marker -, and HPIP-positive tumor cells, and a more significant stimulatory effect on the amount of cleaved caspase-3 apoptosis marker -positive tumor cells Fig.

Furthermore, the lung metastasis of the TXX treatment group was significantly decreased compared with that of the rimonabant group Fig. Taken together, these data suggested that the TXX inhibits breast tumor growth and lung metastasis, and TXX shows a more potent anticancer effect than rimonabant.

B Representative bioluminescent images of animals were taken at the day 0, 7th-, 14th-, and 21st-day postbioluminescent MDA-MB xenografts left panel.

Scale bar: 50 μm. Representative images were taken at the 0, 7th, 14th, and 21st day, respectively left panel. C Proposed model for TXX inhibition of breast cancer growth and metastasis.

TXX decreases HPIP expression, leading to the regulation of inducing cell cycle arrest, apoptosis and inactivation of the metastasis-associated gene, causing growth and metastasis inhibition of breast cancer. The TXX has a more effective inhibition on breast cancer cell proliferation, migration, invasion, and metastasis in vitro and in vivo than rimonabant.

Genome-wide RNA sequencing indicated TXX has more significant effects on the regulation of the expression of genes involved in cancer cell growth and metastasis than rimonabant. Furthermore, compared with rimonabant, the blood-brain barrier permeability of TXX is decreased to avoid psychotropic side effects.

These findings suggest that the TXX can be a candidate drug for inhibition of cancer growth and metastasis. Since HPIP modulates cancer cell growth, invasion, epithelial-mesenchymal transition EMT and metastasis through regulating various genes, including AKT, ERK, FAK, and mTOR signaling [ 13 , 14 , 15 , 25 , 27 ].

Moreover, knockout of HPIP alleviated the effect of TXX on AKT and ERK inhibition, demonstrating the role of HPIP in TXXregulated AKT and ERK signaling. It has also been reported that HPIP interacts with ERα and increasing ERα target genes expression, including pS2 and cathepsin D through activation of MAPK and AKT [ 13 ].

In this paper, we also explored whether TXX treatment affect ERα expression, HPIP-ERα cooperation as well as the modulation of ERα target genes. TXX treatment showed no effect on HPIP-ERα interaction Supplementary Fig. In addition, overexpression of HPIP reversed the inhibitory effect of TXX on pS2 and cathepsin D.

In the last few decades, an increasing number of studies have revealed the aberrant expression of CB1 receptor in numerous types of tumors, which is related to cancer prognosis and disease outcome [ 28 , 29 ]. Several authors have demonstrated that CB1 antagonist rimonabant exerts an anticancer activity in various cancers, including breast cancer [ 30 ] and colon cancer [ 31 , 32 ].

Here, we have also shown that rimonabant suppresses the breast cancer cell proliferation and tumor growth both in vitro and in vivo. However, it was found that rimonabant had neuropsychiatric adverse events, such as alteration of emotional behaviors and cognitive function, which led to its withdrawal from the market [ 19 ].

In this study, we developed TXX, a derivative of rimonabant with a more potent antitumor capacity in inhibiting tumor growth and metastasis than rimonabant both in vitro and in vivo.

Although rimonabant also induced G1 cell cycle arrest, no effects on cell apoptosis and apoptotic-related proteins were observed. In addition, TXX inhibits cancer metastasis via decreasing the phosphorylation and catalytic activity of proteins related to the FAK signal pathway, whereas rimonabant showed a little suppressive effect of cancer metastasis as well as related proteins.

Furthermore, RNA-sequencing analysis also reveals that TXX generally leads to a more significant suppression or stimulation of genes than rimonabant involved in tumor-associated biological processes.

Given the side effects of rimonabant, we detected the BBB permeability of TXX The results indicate that the latter would not exert unwanted psychotropic effects as the three-position of the pyrazole ring of rimonabant is structurally modified with a higher hydrophilic quaternary ammonium salt group.

Indeed, the physicochemical properties predicted by the ADMET predictor 8. Furthermore, TXX was well tolerated in animal studies and showed no side effects on the body weight of mice after 20 days of treatment, indicating that it has fewer side effects.

The physicochemical property prediction also suggests the satisfactory physicochemical properties and low toxicological effects of TXX However, the toxicity and physiochemical properties of TXX in vivo require more detailed investigations. Future studies are also warranted to evaluate the optimized dose and treatment conditions of such an agent as a clinical candidate drug for cancer treatment.

Except for defining tumor-suppressive properties of TXX, it is still necessary to fully understand how TXX regulates gene expression and signaling pathways.

To investigate the molecular mechanisms underlying the function of TXX, we performed RNA-Seq analysis to search for TXXmediated pathways. Gene ontology and KEGG enrichment analysis revealed that TXX modulates genes associated with numerous cellular processes, including DNA replication, cell cycle, apoptosis, focal adhesion, angiogenesis, and energy metabolism.

These processes contribute to cancer development and progression and belong to certain hallmarks of cancer. Other pathways, such as platinum drug resistance, PI3K-Akt signaling pathway, HIF-1 signaling pathway, and glycolysis are downregulated as well.

It has been reported that the aberrant metabolism of cancer cells characterized by high glycolysis facilitates tumor growth and metastasis even in the presence of abundant oxygen. The reprogramming of tumor cell energy metabolism has attracted great interest in terms of its application of a new class of effective anticancer treatment strategies [ 33 , 34 ].

It will be interesting to examine whether TXX regulates cancer metabolism, and further studies will also be conducted to combine TXX with other strategies. Collectively, we developed a novel agent named TXX, which significantly reduces HPIP expression and regulates HPIP downstream targets.

TXX has a more effective inhibition on breast cancer cell proliferation, migration, invasion, and metastasis in vitro and in vivo than rimonabant.

The gene-expression signature also reflects the tumor-suppressive properties of TXX on diverse tumor processes, suggesting HPIP may be an attractive therapeutic target for cancer therapy.

Further investigation is warranted to evaluate the efficacy of TXX as a clinical candidate drug or in combination with other strategies for cancer treatment.

The chemical structures and synthetic schemes for TXX are presented in Supplemental Fig. Detailed synthetic procedures are provided below. The mixture was stirred at room temperature overnight.

The organic phase was combined, washed, evaporated, and purified to afford the title compound 1. All cell lines used in this study were purchased from the American Type Culture Collection ATCC, Manassas, Virginia, USA.

TXX and other novel derivatives were designed and synthesized in the laboratory of Professor Zhibin Zheng at National Engineering Research Center for the Emergency Drug through the chemical modification of rimonabant. Compounds were dissolved in DMSO and diluted in normal saline for cell culture studies.

Brain uptake clearance was estimated using a sample pooling method according to a previous study [ 35 ]. Venous blood was taken from heparin sodium anticoagulant tubes after administration. The drug concentration in plasma and brain tissue samples were measured by LCMS liquid chromatography-mass spectrometry Shimadzu, Japan.

The number of colonies with more than 1. The debris were washed with PBS three times and treated with DMSO or compounds in DMEM high glucose without FBS accompanied with mitomycin C 1 μM treatment.

For cell invasion assay, 10 μl liquid Matrigel BD Biosciences melted on the ice was added dropwise to the upper surface of the transwell chamber Corning. Cancer cells were washed with PBS three times and were added to each well with DMSO or compounds treatment-containing medium at 10, cells per well accompanied with mitomycin C 1 μM treatment.

The number of the invaded cells was counted after taking photographs. Propidium Iodide was then added to the cell suspension. Samples were analyzed by a FACS calibur Flow Cytometer Becton Dickinson. NEBNext ®UltraTM RNA Library Prep Kit for Illumina ® NEB, USA was used to generate a sequencing library.

In short, the mRNA was extracted and then purified from total RNA. Double-stranded cDNA was synthesized with these short fragments as templates. The cDNA library was sequenced on an Illumina HiSeq sequencing platform BerryGenomics.

The gene-expression levels for each transcript of the exon model were estimated as the number of reads per kilobase per million mapped reads RPKM using only uniquely mapped reads in exonic regions.

MDA-MB and ZR cells were collected and suspended in ALDEFLUOR assay buffer. Cells were analyzed on a flow cytometer BD Biosciences. FACS data were analyzed with FlowJo software Treestart. Animal studies were approved by the Institutional Animal Care Committee of Beijing Institute of Biotechnology.

Nude mice were purchased from Vital River Laboratory Animal Technology Beijing and housed in an SPF animal facility. The tumor size was measured at the indicated time using callipers. Images of xenograft mice were obtained using a Xenogen IVIS Luminal Imager once a week.

The slides were placed on a Leica BondMax Immunostainer. Antibodies were optimized with a predetermined staining protocol: Ki67, ; CD31 Rabbit mAb ; HPIP Rabbit mAb, , and cleaved-caspase-3 Asp , Slides were dehydrated and cover-slipped with Cytoseal 60 Richard-Allan Scientific mounting medium.

All in vitro experiments were performed in triplicate and repeated three times. The SPSS software All data generated or analyzed during this study are included in this published article and its supplementary information files.

Updates in the evaluation and management of breast cancer. Article Google Scholar. Thorat MA, Cuzick J. Preventing invasive breast cancer using endocrine therapy. Morrow M, Schnitt SJ, Norton L. Current management of lesions associated with an increased risk of breast cancer.

Filippiadis D, Mavrogenis AF, Mazioti A, Palialexis K, Megaloikonomos PD, Papagelopoulos PJ, et al. Metastatic bone disease from breast cancer: a review of minimally invasive techniques for diagnosis and treatment.

Song M, Bode AM, Dong Z, Lee MH. AKT as a therapeutic target for cancer. Cancer Res. Article CAS Google Scholar. Samatar AA, Poulikakos PI. Targeting RAS—ERK signalling in cancer: promises and challenges. Drug Discov.

Lavoie H, Gagnon J, Therrien M. ERK signalling: a master regulator of cell behaviour, life and fate. Cell Biol. Asati V, Mahapatra DK, Bharti SK. Med Chem. Alzahrani AS. Semin Cancer Biol. Roskoski R Jr. Savoia P, Fava P, Casoni F, Cremona O. Targeting the ERK signaling pathway in melanoma.

Int J Mol Sci. Bramanandam M, Filippo A, Suresh KR, Rakesh K. An inherent role of microtubule network in the action of nuclear receptor. Wang X, Yang Z, Zhang H, Ding L, Li X, Zhu C, et al. The estrogen receptor-interacting protein HPIP increases estrogen-responsive gene expression through activation of MAPK and AKT.

Biochimica et. biophysica acta. Bugide S, David D, Nair A, Kannan N, Samanthapudi VS, Prabhakar J, et al. Hematopoietic PBX-interacting protein HPIP is over expressed in breast infiltrative ductal carcinoma and regulates cell adhesion and migration through modulation of focal adhesion dynamics.

Xu X, Fan Z, Kang L, Han J, Jiang C, Zheng X, et al. Hepatitis B virus X protein represses miRNAa to enhance tumorigenesis. CAS PubMed PubMed Central Google Scholar. Feng Y, Li L, Zhang X, Zhang Y, Liang Y, Lv J, et al. Hematopoietic pre-B cell leukemia transcription factor interacting protein is overexpressed in gastric cancer and promotes gastric cancer cell proliferation, migration, and invasion.

Cancer Sci. Feng Y, Xu X, Zhang Y, Ding J, Wang Y, Zhang X, et al. HPIP is upregulated in colorectal cancer and regulates colorectal cancer cell proliferation, apoptosis and invasion. Khumukcham SS, Samanthapudi VSK, Penugurti V, Kumari A, Kesavan PS, Velatooru LR, et al.

Neuropsychiatric adverse effects of centrally acting antiobesity drugs. CNS Neurosci. Green DR. The coming decade of cell death research: five riddles. Hanahan D, Weinberg RA.

Hallmarks of cancer: the next generation. Steeg PS. Targeting metastasis. Lambert AW, Pattabiraman DR, Weinberg RA. Emerging biological principles of metastasis. Roy-Luzarraga M, Hodivala-Dilke K. Molecular pathways: endothelial cell FAK-A target for cancer treatment.

Thank you for visiting nature. You are Innibit a browser version with limited support for Ihibit. To obtain the best experience, we Inyibit you use a more prolliferation Inhibit cancer cell proliferation date browser or Metabolic support vitamins Inhibit cancer cell proliferation compatibility Inhibit cancer cell proliferation proliferatio Internet Explorer. In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript. Here, we screened compounds inhibiting breast cancer cell proliferation with HPIP fused with green fluorescent protein as a reporter. A novel agent named TXX derived from rimonabant, an antagonist of cannabinoid receptor 1 with anticancer effects, has been discovered to reduce HPIP expression and has greater inhibitory effects on breast cancer cell growth and metastasis in vitro and in vivo than rimonabant. TXX regulates HPIP downstream targets, including several important kinases involved in cancer development and progression e.