Research ArticleExperimental Studies

Effects of Spiro-bisheterocycles on Proliferation and Apoptosis in Human Breast Cancer Cell Lines

LAMIA HAMDAN RAMDANI, OUALID TALHI, NADIA TAIBI, LAETITIA DELORT, CAROLINE DECOMBAT, ARTUR SILVA, KHALDOUN BACHARI, MARIE-PAULE VASSON and FLORENCE CALDEFIE-CHEZET
Anticancer Research December 2016, 36 (12) 6399-6408;

Abstract

Breast cancer is the leading cause of cancer-related death in women worldwide and a critical public health concern. Here we investigated the anticancer potential and effects of low-molecular-weight bridgehead oxygen and nitrogen-containing spiro-bisheterocycles on proliferation and apoptosis of the human breast cancer cell lines MCF-7 and MDA-MB-231. The compounds feature a hydantoin moiety attached to either diazole, isoxazole, diazepine, oxazepine or benzodiazepine via the privileged tetrahedral spiro-linkage. Treatment with compounds spiro [hydantoin-isoxazole] and spiro [hydantoin-oxazepine] resulted in a dose-dependent decrease of cell proliferation and induction of apoptosis in both breast cancer cell lines, whereas spiro [hydantoin-diazepine] was only active against MDA-MB 231. Quantitative reverse transcription polymerase chain reaction analysis showed up-regulation of murine double minute 2 (MDM2), strictly p53-dependent, and detected an increase in expression of pro-apoptotic caspase 3 and BCL2-associated X (BAX) genes in both breast cancer cell lines expressing wild-type and mutant p53. In summary, the results suggest that our compounds promote apoptosis of breast cancer cell lines via p53-dependent and -independent pathways.

Breast cancer is the most common invasive cancer in women and the leading cause of cancer-related death in women worldwide, making it an urgent public health issue (1). Despite intensive research into new chemotherapeutic agents, treatment is still characterized by unwanted side-effects and the spread of drug resistance, and thus falls short of expectations (2). Consequently, it is essential to pursue the drug development effort to treat breast cancer cells.

Many spiro compounds play fundamental roles in biological processes and have demonstrated therapeutic properties. (3), especially the spiro-heterocyclic compounds, which have shown promising results in chemotherapy of various cancer types (4). Spiro compounds are usually a structural system of two rings positioned orthogonally one to the other due to the sp3 hybridization of the central spiro carbon, which can be chemically adjusted by introducing various heterocyclic motifs mimicking those found in biomolecules, such as DNA and proteins, in order to increase their ability to interact with biological systems. This very rare spatial arrangement of spiro-bicyclic scaffolds is characterized by high rigidity, preventing freely-rotating bonds, which can also be configured with several essential functional groups.

Spiro functionality also remains a primary structural tool for creating powerful molecular antitumor and anticancer agents used in latest-generation chemotherapy. Nature provides outstanding spiro structures, as evidenced by the discovery of new spiro-bicyclic triterpenoid models were found to be cytotoxic against breast human cancer cell lines (5). However, the low natural availability of spiro compounds has prompted researchers to build on these molecular templates to optimize novel spiro-based chemical structures capable of significant bioactivity.

In this context, researchers have successfully used tetrapeptide recognition motifs in order to prepare core systems of 5.5 spiro-oxindoles (Figure 1A) and 6.5.5 spiro-bicyclic lactams (Figure 1B) capable of affecting protein–protein interactions for use as an efficient strategy in cancer therapy (6). Various spiro-isoxazolidine (Figure 1C) derivatives were prepared and tested for their anticancer activity against human cancer cell lines. Moreover, several 4-bromo spiro-isoxazoline structures have also shown antiproliferative effects against breast and prostate cancer cell lines (7).

The transcription factor tumor suppressor p53 is one of the main mediators regulating the cell cycle and induction of apoptosis in response to cellular damage (9). p53 regulation in the cell is essentially based on its negative regulator murine double minute 2 (MDM2). Targeting p53–MDM2 interaction by using appropriate molecules offers an attractive strategy for p53 activation (8) and a promising approach for new anticancer therapies (10).

Previous studies have reported that spiro compounds are potent antagonists of the p53–MDM2 interaction (6). Given these biorelevant properties, spiro compounds make very attractive targets for cancer treatment screening. Here we provide an account of the evaluation of our previously reported small molecules containing oxygen and nitrogen spiro-bisheterocycles in terms of effects on in vitro proliferation and apoptosis of human breast cancer cell lines (MCF-7 and MDA-MB231). The wider aim is to identify novel low-molecular-weight, easily-accessible synthetic spiro compounds for cancer chemotherapy.

Materials and Methods

Chemistry. Spiro-bisheterocyclic compounds 2-7 (Table I; Figure 2) were chemically synthesized following our previously described procedure (11). Their preparation is based on the action of bisnucleophiles such as methylhydrazine, hydroxylamine, ethylenediamine, hydroxyethyleneamine and ortho-phenylenediamine on the key spiro[chromanone-hydantoin] (1) dyad, which results in a spiro-to-spiro ring transformation of the chromanone residue into new substituted spiro[hydantoin-diazole] (2), spiro[hydantoin-isoxazole] (3), spiro[hydantoin-diazepine] (4-5), spiro[hydantoin-oxazepine] (6) and spiro[hydantoin-benzodiazepine] (7) (Figure 2). During these chemical reactions, the hydantoin cycle is preserved, as confirmed by relevant 2D nuclear magnetic resonance and single-crystal X-ray diffraction studies. Our spiro-bisheterocycles (2-7) were produced in optimal 42-67% yields after chromatographic purification and recrystallization, thus affording high purity as checked by TLC showing a single spot.

Cell culture and treatment. MCF-7 and MDA/MB 231 human breast adenocarcinoma cell lines were purchased from the American Type Culture Collection (Molsheim, France). MCF-7 and MDA-MB-231 were grown in RPMI-1640 and L-15 medium (GIBCO, Invitrogen, Saint-Aubin, France), respectively. RPMI-1640 medium was supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Eurobio, Courtaboeuf, France), 1% L-glutamine (2 mM) (GIBCO, Invitrogen, Saint-Aubin, France), 0.5% gentamycin (50 mg/ml) (Fisher Scientific, Strasbourg, France) and 0.05% insulin (100 UI/ml). Fluoresceinisothiocyanate (FITC) conjugated to annexin V was purchased from BioLegend (San Diego, CA, USA) and propidium iodide (PI) was purchased from Biotium (Hayward, CA, USA). A 10 mM stock solution of compounds 2-7 was prepared in dimethylsulfoxide (DMSO) and stored at 4°C. All compounds were added to cells at different concentrations. The DMSO concentration of controls was <0.1% (v/v) and 1 μM doxorubicin was used as a positive control.

View this table:
Table I.

Structures and experimental data of spiro-bisheterocycles 2-7 (11).

Cell proliferation assay. Cell proliferation was assessed by resazurin assay. Human breast adenocarcinoma cell lines (1×104/well) were cultured in 96-well plates with complete media for 24 h. At the end of incubation, cells were treated with compounds 2-7 at concentrations of 0, 10, 25, 50 and 100 μM for 72 h at 37°C under a 5% CO2 atmosphere without replacing the medium. Control cells and positive control cells were treated with 0.1% DMSO and 1 μM doxorubicin, respectively. After incubation, 200 μl of a 25 μg/ml solution of resazurin in medium was added to each well. Plates were incubated for 2 h at 37°C in a humidified atmosphere containing 5% CO2. Fluorescence was then measured on an automated 96-well plate reader (Fluorskan Ascent FL; Thermo Fisher Scientific, Wilmington, NC, USA) at 530 nm excitation wavelength and 590 nm emission wavelength. Under these conditions, fluorescence (OD value) was proportional to the number of living cells in the well. Cell proliferation assays were performed at least six times for each cell line (in replicates of six wells per concentration tested) (12).

Figure 1.
Figure 1.

Synthetic spiro-bicyclic compounds as anticancer agents: A: 5.5 spiro-oxindoles; B: 6.5.5 spiro-bicyclic lactams; and C: spiro-isoxazolidine.

Figure 2.
Figure 2.

Reaction pathway for the synthesis of spiro-bisheterocycles 2-7 (11). RT: Room temperature; DBU: 1,8-diazabicyclo[5.4.0]undec-7-ene; THF: tetrahydrofuran.

DNA quantification using Hoechst 33342. 96-Well plates containing treated and control cells were taken out of freezer storage at −20°C and left to defrost for 1 h at room temperature. Then, 100 μl of 0.01% sodium dodecyl sulfate was added to enable the DNA to come into contact with the Hoechst solution. After 1 h of incubation, plates were put back in the freezer at −80°C for 1 h to trigger thermal shock. After defrosting for 2 h, 100 μl of Hoechst solution 33342 (30 μg ml−1) was added to each well. Plates were placed under agitation for 1 h shielded from light. The reading of the fluorescence was made by Fluorskan Ascent FL (Thermo Fisher Scientific). Hoechst 33342 dye is a fluorescent nucleic acid which exhibits a maximum emission at 460 nm when bound specifically to double-stranded DNA. Data represent viable cells.

Annexin V–FITC/PI apopstosis assay. MCF-7 and MDA-MB 231 tumor cells (105 cells/well) were treated with 100 μM of compounds 3, 4 and 6 for 72 h at 37°C andwith 5% CO2, then washed with phosphate-buffered saline, recovered by centrifugation at 1,000 × g for 5 min at room temperature (RT; 25°C), and resuspended in 40 μl of annexin V binding buffer (140 mm NaCl, 10 mm HEPES/NaOH, 2.5 mm CaCl2). The suspension was stained with 5 μl of annexin V-FITC and 5 μl of PI, then incubated for 15 min at RT in the dark. To the sample was added 250 μl of 1×phosphate-buffered saline, this was then centrifuged at 1,000 × g for 5 min at RT, and cells were resuspended in 50 μl of annexin V binding buffer. Stained cells were then analyzed on a Cellometer K2 image cytometer (Nexcelom Bioscience, Lawrence, MA, USA).

View this table:
Table II.

Primer sequences.

Isolation of RNA and real-time polymerase chain reaction. Total RNA was extracted from untreated and treated breast cancer cell lines, MCF-7 and MDA-MB-231, using Trizol and quantified on a NanoDrop spectrophotometer (Nanodrop 2000; Thermo Scientific, Waltham, MA, USA). Purity was estimated by 260/280 nm absorbance ratio. cDNA was synthesized using the High-Capacity cDNA Reverse Transcription kit (Applied Biosystems, Courtaboeuf, France). Quantitative real-time PCR analysis was performed using a SYBR Green PCR Master Mix (Applied Biosystems, Courtaboeuf, France) as follows: first step at 50°C for 2 min, denaturation step at 95°C for 10 min, then 40 cycles at 95°C for 15 s and 60°C for 1 min. In total, 10 ng of cDNA was added to 18 μl of reaction containing primers. Relative expression levels of p53, MDM2 and BCL2-associated X (BAX) were calculated using the 2−ΔΔCt method. Experiments were repeated in triplicate. The primer sequences used are listed in Table II.

Data analysis. Data are expressed as the mean±SEM of multiple experiments. Statistical significance was determined by Student's t-test using SPSS software (ver. 18.0; SPSS Inc., Chicago, IL, USA).

Results

Effects of spiro-bisheterocycles 2-7 on MCF-7 and MDA-MB231 cell proliferation. In order to determine the impact of spiro-bisheterocycles 2-7 on cell proliferation of human breast cancer cell lines, MCF-7 and MDA-MB 231 cells were treated with compounds 2-7 at different concentrations from 10 to 100 μM for 72 h. Cell proliferation was assessed after 2 h of incubation with resazurin solution. Proliferation of MCF-7 human breast cancer cell line was significantly reduced with compounds 3 and 6, with The half-maximal inhibitory concentration (IC50) corresponding to 42.3 μM (17.4 μg/ml) and 66.3 μM (30.8 μg/ml) (Figure 3), respectively. We also observed significant inhibition of MDA-MB-231 cells (Figure 3) up to 67.9 μM (28.5 μg/ml) for 3 and 97.1 μM (42.7 μg/ml) for 6. Proliferation of MDA-MB 231 cells was also significantly reduced with compound 4, with IC50 of 44.6 μM (19.6 μg/ml), but not of MCF-7 cells. On the other hand, treatment with compounds 2, 5 and 7 did not induce a significant decrease in cell proliferation in the two breast cancer cell lines. At a 100 μM, compound 3 was the most effective, inducing a major suppressive effect of 98.15% and 97.02% against MCF-7 and MDA-MB 231 cells, respectively.

Hoechst staining confirmed results of the resazurin assay and showed significant impact of compounds 3 and 6 on MCF-7 and MDA-MB-231 tumor cell proliferation (Figure 4), with similar IC50. Compound 4 induced tumor inhibition at an IC50 of 42 μM inMDA-MB-231 cells.

Effects of spiro-bisheterocycles 3, 4 and 6 on apoptosis. In order to determine the apoptotic efficacy of the spiro-bisheterocycles, cells were treated with 100 μM of 3, 4 and 6 for 72 h (Figure 5), then collected and subjected to Cellometer annexin V-FITC/PI apoptosis assay. Annexin V-FITC−/PI− flagged viable cells, annexin V-FITC+/PI− flagged early-stage apoptotic cells, and annexin V-FITC+/PI+ flagged late-stage apoptotic cells.

Analysis showed a sharp induction of apoptosis by compounds 3 and 6 in MCF-7 cells and by 3, 4 and 6 in MDA-MB-231 cells (Figure 5A). The rates of early and late apoptosis were 9.5%, 85.4% and 71.3% in MCF-7 for control, and compounds 3 and 6, respectively, and 5.9%, 88.1%, 79.4% and 66.4% in MDA-MB-231 cells for control, and compounds 3, 4 and 6, respectively (Figure 5B).

Effects of spiro-bisheterocycles 3, 4 and 6 on p53, MDM2, BAX and caspase 3 expression. In an effort to investigate whether spiro compounds 3, 4 and 6 induce apoptosis of breast cancer cell lines by targeting p53–MDM2 interaction, we used quantitative reverse transcription-polymerase chain reaction to analyze the wild-type p53, MDM2, BAX and caspase 3 gene expression levels. Results for MCF-7 breast cancer cells show that compound 3 more clearly increased the expression of wild-type p53 (6.707-fold vs. control) than did compound 6 (3.275-fold vs. control) (Figure 6). We also found that compounds 3 and 6 induced up-regulation of MDM2 in MCF-7 cells at levels of 4.282 and 2.658, respectively. However, in MDA-MB-231 p53-mutant breast cancer cells, no significant changes in the levels of p53 and MDM2 were observed with compounds 3, 4 and 6, indicating that overexpression of MDM2 is under control of p53. Compared to levels in untreated control MCF-7 cells, compounds 3 and 6 led to 6.738- and 3.532-fold higher BAX and 6.498- and 2.703-fold higher caspase 3 gene expression, respectively. Interestingly, similar results were obtained in MDA-MB-231 cells, where an increase of the expression of caspase 3 and BAX genes was also noted with compounds 3, 6 and, even with compound 4. This demonstrates the capacity of our compounds to induce apoptosis independently of p53 status.

Figure 3.
Figure 3.

Effect of compounds 2-7 on the in vitro growth and proliferation of human breast cancer cells. MCF-7 and MDA-MB231 cell line were either untreated, treated with 1 μM doxorubicin (Dox) or treated with 10, 25, 50 and 100 μM of compounds 2-7 for 72 h as described in the Materials and Methods section. Proliferation was assessed using resazurin on an automated 96-well Fluoroskan Ascent FL. Columns show means±SD, n=6. *Significantly different at p<0.05.

Figure 4.
Figure 4.

Inhibitive effect of compounds 3, 4 and 6 on growth of cancer cell lines MCF-7 and MDA-MB-231. *Significantly different at p<0.05.

Discussion

Tumor suppressor p53 plays a key role in the regulation of cell cycle and apoptosis (13-15). We previously reported that p53 inhibition by its negative regulator MDM2 is involved in the onset of breast cancer (16, 17). An approach based on restoring p53 activity by inhibiting p53–MDM2 interaction could lead to tumor suppression and thus be a promising strategy for future control of breast cancer (18, 19). Many spiro compounds possess very promising biological and pharmacological activities, especially antitumor and antimicrobial properties (4, 20, 21).

These specific, potent, non-peptide small-molecule inhibitors are reported to mimic the α-helix recognition motif of the p53–MDM2 complex (22), thereby efficiently reactivating p53 tumor-suppressor activity and consequently inducing a p53-mediated signaling pathway that will culminate in cell death by apoptosis (23). Our results clearly showed that the spiro-heterocyclic compounds substituted by isoxazole (3), diazepine (4) and oxazepine (6) significantly reduced the proliferation of MCF-7 and MDA-MB-231 cells in a dose-dependent manner. These results were consistent with other studies demonstrating the cytotoxicity of spiro-bicyclic compounds in human cancer cell lines (5, 24). Our data are further supported by another report which demonstrated similar in vivo anticancer potential of a spiro-heterocyclic compound on the development on MCF-7 human breast cancer cells (25). The antiproliferative effect was more pronounced with spiro-isoxazole (3). Structurally, the presence of the isoxazole heterocyclic unit is likely tightly related to these clear antiproliferative effects, as already reported for other spiro-models containing the isoxazole motif (7). It was also observed that the presence of oxazepine substituent demonstrated good activity against breast cancer cells (26), thus explaining the antiproliferative effect observed with compound 6. Furthermore, the significant antiproliferative activity of compound 4 could stem from the presence of a diazepine molecule in the structure, since diazepine inhibits the adenosine A2B receptor involved in the development of tumors and proliferation (27). In addition, a previous study reported that adenosine A2B receptors are expressed at high levels in the estrogen-negative MDA-MB-231 cell line but not expressed in the estrogen-positive MCF-7 cell line, which could explain why the antiproliferative effect of compound 4 was observed here only in MDA-MB-231 cells (28). Moreover, the presence of cyclohexyl fragments in these three compounds might also contribute to their antiproliferative activity (29), allowing better understanding of why compounds 5 and 7 had no effect.

Figure 5.
Figure 5.

Drug-induced apoptosis in breast cancer cell lines MCF-7 and MDA-MB-231 at 72 h of incubation with spiro-bisheterocyclic compounds. A: Bright-field (top row), and fluorescent images of annexin V-fluorescein isothiocyanate (FITC) (middle row) and propidium iodide (PI) (bottom row) staining. The images show significant induction of apoptosis with compounds 3 and 6 in MCF-7cells, while strong apoptosis was observed with compounds 3, 4 and 6 in MDA-MB-231 cells. B: Quantitative results of percentage of viable, and early apoptotic and late apoptotic cells after treatment with compounds. *Significantly different at p<0.05.

Apoptosis plays a key role in the maintenance of homeostasis (30) and is considered a primary form of cancer cell death (31). This pathway results in DNA fragmentation, degradation of cytoskeletal and nuclear proteins, formation of apoptotic bodies, and finally uptake by phagocytic cells (32). Recent reports indicate that most spiro compounds induce apoptosis of cancer cells (33, 34), but especially spiro-isoxazolidine derivatives (35). Here, we performed annexin V-FITC/PI apoptosis assays and demonstrated that our spiro-bisheterocyclic compounds induced apoptosis of breast cancer cell lines. Annexin V is a protein that interacts strongly and specifically with phosphatidylserine. During early apoptosis, phosphatidylserine translocates to the external leaflet and can be used for the detection of apoptosis (36). Analysis using Cellometer K2 image cytometry indicated that compounds 3, 4 and 6 induced a large proportion of breast cancer cell death by apoptosis in vitro. The strong effect was observed with compound 3 in both breast cancer cell lines.

As previously demonstrated (18), the most likely mechanism by which spiro compounds initiate apoptosis of wild-type p53 breast cancer cells is by inhibition and disruption of the p53–MDM2 interaction, leading to accumulation of p53 and up-regulation of MDM2. The tumor suppressor p53 binds to DNA and mediates transcriptional activation which ultimately overexpress BAX and promote p53-dependent cell apoptosis (37, 38). The pro-apoptotic BAX protein is one of the most important regulators of the intrinsic pathway of apoptosis (39). Excess of BAX protein leads to formation of BAX–BAX homodimers, that stimulate release of cytochrome c from mitochondria and activate caspase-3, then inducing apoptosis (40).

Figure 6.
Figure 6.

Results of polymerase chain reaction detection. p53, murine double minute 2 (MDM2), BCL2–associated X (BAX) and caspase-3 genes expression in MCF-7 and MDA-MB-231 cells following treatment with 100 μM of compounds 3, 4 and 6 for 72 h. *Significantly different at p<0.05.

Quantitative reverse transcription-polymerase chain reaction data support these findings and showed an increase of wild-type p53 expression in MCF-7 cells, accompanied by up-regulation of MDM2, with treatment of compound 3 and 6. We extended this study by demonstrating that spiro compounds 3 and 6 enhance expression of caspase-3 and BAX in MCF-7 wild-type p53 breast cancer cells, and consequently induce apoptosis. At the same time, in order to determine whether our spiro compounds induce apoptosis regardless of cell p53 status, we also employed MDA-MB-231 cells which harbor mutant p53. Treatment with compounds 3, 4 and 6 had no significant effect on expression of p53 or MDM2 in cancer cells lacking wild-type p53 but increased caspase-3 and BAX expression. These results correlate with a previous study revealing induction of apoptosis through p53-independent pathways (41).

Figure 7.
Figure 7.

Proposed mechanisms of apoptosis of breast cancer MCF-7 and MDA-MB-231 cells induced by spiro-compounds 3, 4 and 6.

In summary (Figure 7), this study brings the first demonstration that new small spiro-bisheterocyclic molecules can sensitize breast cancer cells to apoptosis by targeting p53–MDM2 interaction. We also highlighted that these spiro compounds promote apoptosis via p53-independent pathway(s), suggesting that these compounds represent interesting candidates as therapeutic targets for the treatment of breast cancer.

Acknowledgements

The Authors thank the Unité de Nutition Humaine UMR 1019 INRA-UdA – Equipe ECREIN (France) and the University of Aveiro, Fundaćão para a Ciência e a Tecnologia (Portugal), EU, QREN, FEDER, COMPETE, for funding this biological and organic chemistry research. They also thank Algeria's General Directorate for Scientific Research and Technological Development (DGRSDT) for financial support.

  • Received October 12, 2016.
  • Revision received November 2, 2016.
  • Accepted November 9, 2016.

References

  1. ↵
    1. Ferlay J,
    2. Shin HR,
    3. Bray F,
    4. Forman D,
    5. Mathers C,
    6. Parkin DM
    : Estimates of worldwide burden of cancer in 2008. Int J Cancer 127: 2893-2917, 2010.
    OpenUrlCrossRefPubMed
  2. ↵
    1. Rokhlin OW,
    2. Gudkov AV,
    3. Kwek S,
    4. Glover RA,
    5. Gewies AS,
    6. Cohen MB
    : p53 is involved in tumor necrosis factor-alpha-induced apoptosis in the human prostatic carcinoma cell line LNCaP. Oncogene 19: 1959-1968, 2000.
    OpenUrlCrossRefPubMed
  3. ↵
    1. El-Hashash MA,
    2. Rizk SA,
    3. Atta-Allah SR
    : Synthesis and regioselective reaction of some unsymmetrical heterocyclic chalcone derivatives and spiro heterocyclic compounds as antibacterial agents. Molecules 20: 22069-22083, 2015.
    OpenUrlPubMed
  4. ↵
    1. Miao Y,
    2. Juan X,
    3. Jing-Jing Z,
    4. Jun-Lin H,
    5. Yun Z,
    6. Hui F,
    7. Yang GX,
    8. Xia G,
    9. Hu JF
    : Leonurusoleanolides E–J, minor spirocyclic triterpenoids from Leonurus japonicus fruits. J Nat Prod 77: 178-182, 2014.
    OpenUrl
  5. ↵
    1. Moujir LM,
    2. Seca AM,
    3. Araujo L,
    4. Silva AM,
    5. Barreto MC
    : A new natural spiro heterocyclic compound and the cytotoxic activity of the secondary metabolites from Juniperus brevifolia leaves.Fitoterapia 82: 225-229, 2011.
    OpenUrlPubMed
  6. ↵
    1. Ding K,
    2. Lu Y,
    3. Nikolovska-Coleska Z,
    4. Qiu S,
    5. Ding Y,
    6. Gao W,
    7. Stuckey J,
    8. Krajewsk,i K,
    9. Roller PP,
    10. Tomita Y,
    11. Parrish DA,
    12. Deschamps JR,
    13. Wang S
    : Structure-based design of potent non-peptide MDM2 inhibitors. J Am Chem Soc 127: 10130-10131, 2005.
    OpenUrlCrossRefPubMed
  7. ↵
    1. Das P,
    2. Omollo AO,
    3. Sitole LJ,
    4. McClendon E,
    5. Valente EJ,
    6. Raucher D
    : Synthesis and investigation of novel spiro-isoxazolines as anti-cancer agents. Tetrahedron Lett 56: 1794-1797, 2015.
    OpenUrlPubMed
  8. ↵
    1. Vassilev LT
    : MDM2 inhibitors for cancer therapy. Trends Mol Med 13: 23-31, 2007.
    OpenUrlCrossRefPubMed
  9. ↵
    1. Yin Y,
    2. Stephen CW,
    3. Luciani MG,
    4. Fahraeus R
    : P53 stability and activity is regulated by MDM2-mediated induction of alternative p53 translation products. Nat Cell Biol 4: 462-467, 2002.
    OpenUrlCrossRefPubMed
  10. ↵
    1. Wade M,
    2. Li YC,
    3. Wahl GM
    : MDM2, MDMX and p53 in oncogenesis and cancer therapy. Nat Rev Cancer 13: 83-96, 2013.
    OpenUrlCrossRefPubMed
  11. ↵
    1. Talhi O,
    2. Pinto DCGA,
    3. Paz FAA,
    4. Hamdi M,
    5. Silva AMS
    : Synthesis and ring transformation ofoxygen and nitrogen spiro-bisheterocycles. Synlett 26: 167-172, 2015.
    OpenUrl
  12. ↵
    1. Dubois V,
    2. Jardé T,
    3. Delort L,
    4. Billard H,
    5. Bernard-Gallon D,
    6. Berger E,
    7. Geloen A,
    8. Vasson MP,
    9. Caldefie-Chezet F
    : Leptin induces a proliferative response in breast cancer cells but not in normal breast cells. Nutr Cancer 66: 645-655, 2014.
    OpenUrlPubMed
  13. ↵
    1. El-Deiry WS
    : Regulation of p53 downstream genes. Semin Cancer Biol 8: 345-357, 1998.
    OpenUrlCrossRefPubMed
    1. Levine AJ
    : p53, the cellular gate keeper for growth and division. Cell 88: 323-331, 1997.
    OpenUrlCrossRefPubMed
  15. ↵
    1. Vogelstein B,
    2. Lane D,
    3. Levine AJ
    : Surfing the p53 network. Nature 408: 307-310, 2000.
    OpenUrlCrossRefPubMed
  16. ↵
    1. Rayburn E,
    2. Zhang R,
    3. He J,
    4. Wang H
    : MDM2 and human malignancies: expression, clinical pathology, prognostic markers, and implications for chemotherapy. Curr Cancer Drug Targets 5: 27-41, 2005.
    OpenUrlCrossRefPubMed
  17. ↵
    1. Vassilev LT,
    2. Vu BT,
    3. Graves B,
    4. Carvajal D,
    5. Podlaski F,
    6. Filipovic Z,
    7. Kong N,
    8. Kammlott U,
    9. Lukacs C,
    10. Klein C,
    11. Fotouhi N,
    12. Liu EA
    : In vivo activation of the p53 pathway by small-molecule antagonists of MDM2. Science 303: 844-848, 2004.
    OpenUrlAbstract/FREE Full Text
  18. ↵
    1. Shangary S,
    2. Qin D,
    3. McEachern D,
    4. Liu M,
    5. Miller RS,
    6. Qiu S,
    7. Nikolovska-Coleska Z,
    8. Ding K,
    9. Wang G,
    10. Chen J,
    11. Bernard D,
    12. Zhang J,
    13. Lu Y,
    14. Gu Q,
    15. Shah RB,
    16. Pienta KJ,
    17. Ling X,
    18. Kang S,
    19. Guo M,
    20. Sun Y,
    21. Yang D,
    22. Wang S
    : Temporal activation of p53 by a specific MDM2 inhibitor is selectively toxic to tumors and leads to complete tumor growth inhibition. Proc Natl Acad Sci USA 105: 3933-3938, 2008.
    OpenUrlAbstract/FREE Full Text
  19. ↵
    1. Shangary S,
    2. Wang S
    : Targeting the MDM2–p53 interaction for cancer therapy. Clin Cancer Res 14: 5318-5324, 2008.
    OpenUrlAbstract/FREE Full Text
  20. ↵
    1. Rompaey KV,
    2. Ballet S,
    3. Tömböly C,
    4. Wachter RD,
    5. Vanommeslaeghe K,
    6. Biesemans M,
    7. Willem R,
    8. Tourwé DA
    : Synthesis and evaluation of the β-turn properties of 4-amino-1,2,4,5-tetrahydro-2-benzazepin-3-ones and of their spirocyclic derivative.Eur J Org Chem 13: 2899-2911, 2006.
    OpenUrl
  21. ↵
    1. Rosy P,
    2. Manabendra P,
    3. Behera AK,
    4. Mishra BK,
    5. Behera RK
    : A synthon approach to spiro compounds. Tetrahedron 62: 779-828, 2006.
    OpenUrl
  22. ↵
    1. Somu RV,
    2. Johnson RL
    : Synthesis of pipecolic acid-based spiro bicyclic lactam scaffolds as β-turn mimics. J Org Chem 70: 5954-5963, 2005.
    OpenUrlPubMed
  23. ↵
    1. Shangary S,
    2. Wang S
    : Small-molecule inhibitors of the MDM2–p53 protein–protein interaction to reactivate p53 function: a novel approach for cancer therapy. Annu Rev Pharmacol Toxicol 49: 223-241, 2009.
    OpenUrlCrossRefPubMed
  24. ↵
    1. Manner S,
    2. Oltner VT,
    3. Oredsson S,
    4. Ellervik U,
    5. Frejd T
    : Spiro-bicyclo[2.2.2]octane derivatives as paclitaxel mimetics. Synthesis and toxicity evaluation in breast cancer cell lines. Org Biomol Chem 7: 7134-44, 2013.
    OpenUrl
  25. ↵
    1. Chin YW,
    2. Salim AA,
    3. Su BN,
    4. Mi Q,
    5. Chai HB,
    6. Riswan S,
    7. Kardono LB,
    8. Ruskandi A,
    9. Farnsworth NR,
    10. Swanson SM,
    11. Kinghorn AD
    : Potential anticancer activity of naturally occurring and semisynthetic derivatives of aculeatins A and B from Amomum aculeatum. J Nat Prod 71: 390-395, 2008.
    OpenUrlPubMed
  26. ↵
    1. Dhanya S,
    2. Ranjitha C,
    3. Rama M,
    4. KSR P
    : Oxazepine derivative as an antitumor agent and SNAIL1 inhibitor against human colorectal adenocarcinoma. Int J Innov Res Sci Eng Technol 3(8): 15357-15363, 2014.
    OpenUrl
  27. ↵
    1. Hoffmann K,
    2. Xifró RA,
    3. Hartweg JL,
    4. Spitzlei P,
    5. Meis K,
    6. Molderings GJ,
    7. von Kügelgen I
    : Inhibitory effects of benzodiazepines on the adenosine A(2B) receptor mediated secretion of interleukin-8 in human mast cells. Eur J Pharmacol 700: 152-158, 2013.
    OpenUrlPubMed
  28. ↵
    1. Panjehpour M,
    2. Castro M,
    3. Klotz KN
    : Human breast cancer cell line MDA-MB-231 expresses endogenous A2B adenosine receptors mediating a Ca2+ signal. Br J Pharmacol 145: 211-218, 2005.
    OpenUrlCrossRefPubMed
  29. ↵
    1. Bhat MA,
    2. Al-Dhfyan A,
    3. Naglah AM,
    4. Khan AA,
    5. Al-Omar MA
    : Lead optimization of 2-cyclohexyl-N-[(Z)-(3-methoxyphenyl/3-hydroxyphenyl) methylidene]hydrazine-carbothioamides for targeting the HER-2 overexpressing breast cancer cell line SKBr-3. Molecules 20: 18246-18263, 2015.
    OpenUrlPubMed
  30. ↵
    1. Norbury CJ,
    2. Hickson ID
    : Cellular responses to DNA damage. Annu Rev Pharmacol Toxicol 41: 367-401, 2001.
    OpenUrlCrossRefPubMed
  31. ↵
    1. Elmore S
    : Apoptosis: A review of programmed cell death. Toxicol Pathol 35: 495-516, 2007.
    OpenUrlCrossRefPubMed
  32. ↵
    1. Martinvalet D,
    2. Zhu P,
    3. Lieberman J
    : Granzyme A induces caspase-independent mitochondrial damage, a required first step for apoptosis. Immunity 22: 355-370, 2005.
    OpenUrlCrossRefPubMed
  33. ↵
    1. Molvi KI,
    2. Haque N,
    3. Awen BZS,
    4. Zameeruddin M
    : Synthesis of spiro compounds as medicinal agents; new opportunities for drug design and discovery. World J Pharm Sci 3: 536-563, 2014.
    OpenUrl
  34. ↵
    1. Yu B,
    2. Shi XJ,
    3. Qi PP,
    4. Yu DQ,
    5. Liu HM
    : Design, synthesis and biological evaluation of novel steroidal spiro-oxindoles as potent antiproliferative agents. J Steroid Biochem Mol Biol 141: 121-134, 2014.
    OpenUrlPubMed
  35. ↵
    1. Khazir J,
    2. Singh PP,
    3. Reddy DM,
    4. Hyder I,
    5. Shafi S,
    6. Sawant SD,
    7. Chashoo G,
    8. Mahajan A,
    9. Alam MS,
    10. Saxena AK,
    11. Arvinda S,
    12. Gupta BD,
    13. Kumar HM
    : Synthesis and anticancer activity of novel spiro-isoxazoline and spiro-isoxazolidine derivatives of α-santonin. Eur J Med Chem 63: 279-289, 2013.
    OpenUrlPubMed
  36. ↵
    1. Sean RS,
    2. Arthur CM,
    3. Ju T,
    4. Rodrigues LC,
    5. Riul TB,
    6. Dias-Baruffi M,
    7. Miner J,
    8. McEver RP,
    9. Cummings RD
    : Galectin-1 induces reversible phosphatidylserine exposure at the plasma membrane. Mol Biol Cell 20: 1408-1418, 2009.
    OpenUrlAbstract/FREE Full Text
  37. ↵
    1. Li B,
    2. Dou QP
    : BAX degradation by the ubiquitin/proteasome-dependent pathway: involvement in tumor survival and progression. Proc Natl Acad Sci USA 97: 3850-3855, 2000.
    OpenUrlAbstract/FREE Full Text
  38. ↵
    1. Scheffner M,
    2. Werness BA,
    3. Huibregtse JM,
    4. Levine AJ,
    5. Howley PM
    : The E6 oncoprotein encoded by human papillomavirus types 16 and 18 promotes the degradation of p53. Cell 63: 1129-1136, 1990.
    OpenUrlCrossRefPubMed
  39. ↵
    1. Williams GT,
    2. Smith CA
    : Molecular regulation of apoptosis: genetic controls on cell death. Cell 74: 777-779, 1993.
    OpenUrlCrossRefPubMed
  40. ↵
    1. Kurabayashi A1,
    2. Furihata M,
    3. Matsumoto M,
    4. Ohtsuki Y,
    5. Sasaguri S,
    6. Ogoshi S
    . Expression of BAX and apoptosis-related proteins in human esophageal squamous cell carcinoma including dysplasia. Mod Pathol 14: 741-747, 2001.
    OpenUrlPubMed
  41. ↵
    1. Lanni JS,
    2. Lowe SW,
    3. Licitra EJ,
    4. Liu JO,
    5. Jacks T
    : p53-independent apoptosis induced by paclitaxel through an indirect mechanism. Proc Natl Acad Sci USA 94: 9679-9683, 1997.
    OpenUrlAbstract/FREE Full Text
Back to top

In this issue

Print
Download PDF
Article Alerts
Email Article
Citation Tools
Reprints and Permissions
Effects of Spiro-bisheterocycles on Proliferation and Apoptosis in Human Breast Cancer Cell Lines
LAMIA HAMDAN RAMDANI, OUALID TALHI, NADIA TAIBI, LAETITIA DELORT, CAROLINE DECOMBAT, ARTUR SILVA, KHALDOUN BACHARI, MARIE-PAULE VASSON, FLORENCE CALDEFIE-CHEZET
Anticancer Research Dec 2016, 36 (12) 6399-6408;
Twitter logo Facebook logo Mendeley logo

Jump to section

Keywords