Cucurbitacin B Restored Cisplatin Sensitivity of Ovarian Cancer Cells by Altering Fatty Acid Synthase and LRP-130 Protein Expression

Fardous El-Senduny, F.^1,3, Farid Badria, A.², Ahmed EL-Waseef, M.³, Eduardo Callegari, A.⁴ & Fathi Halaweish^1*

¹Chemistry and Biochemistry Department, South Dakota State University, Brookings, USA
²Pharmacognosy Department, Faculty of Pharmacy, Mansoura University, Egypt
³Chemistry Department, Faculty of Science, Mansoura University, Egypt
⁴Division of Basic Biomedical Sciences, Sanford School of Medicine, The University of South Dakota, USA

*Correspondence to: Dr. Fathi Halaweish, Chemistry and Biochemistry Department, South Dakota State University, Brookings, USA.

Copyright © 2018 Fathi Halaweish, et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Introduction
Ovarian cancer is the fifth leading cause to the cancer-related mortality in women. In 2016, there will be 22,280 new cases of ovarian cancer diagnosed and 14,240 women will die of ovarian cancer in the United States. The five-year survival rate is 46.2% and the mortality rates for ovarian cancer have declined only slightly in the forty years [1,2]. This short-term survival is due to the lack of the early detection methods. The first line of chemotherapy treatment is platinum-based drugs such as cisplatin and carboplatin. Most patients suffer from cancer recurrence and became resistant to the chemotherapy regimen. The resistance is due to the increase in drug efflux, detoxification of the drug, high tolerance to sub lethal DNA damage or the capability to repair DNA damage [3]. Till now, research still search for new key in resistance of ovarian cancer to chemotherapy, which will guide the selection of specific inhibitor or combination therapy.

Cucurbitacins are highly oxidized tetracyclic triterpenoid. Cucurbitacin B and D are most dominant types in the plant followed by cucurbitacin E, G, H and I. They are used in Chinese remedies for hepatitis, inflammation and cancer treatment. Cucurbitacin B is the most studied one followed by cucurbitacin E [4-6]. Cucurbitacin B shows anti-proliferative activity against wide range of cancer cell lines such as breast, lung, glioblastoma, hepatic, pancreatic, colon, central nervous system, oral, ovarian and leukemia [7-17]. Moreover, cucurbitacin B increased the cytotoxicity of different chemotherapeutic agents including methotrexate toward osteosarcoma [18], cisplatin against cutaneous squamous cell carcinoma cell lines and laryngeal squamous cell carcinoma cells [19,20], gemcitabine against pancreatic cancer [21,22] and docetaxel in laryngeal cancer [23]. Also cucurbitacin B sensitized the breast cancer cells to the radiotherapy [24]. This sensitization effect of cucurbitacin B attributed to the cell cycle arrest, cytoskeleton alteration [16,25,26], inhibition of signal transducer and activator transcription 3 (STAT-3) [17,18,20,22,27], inactivation of mitogen activated protein kinase pathway [7]and reducing the glutathione content [28]. Recently, we have showed that cucurbitacin B is able to restore the sensitivity of cisplatin-resistant ovarian cancer cells (A2780CP) to cisplatin cytotoxicity [9]. This sensitization effect of cucurbitacin B was found to be due to the inhibition of ERK and STAT-3 phosphorylation, a decrease in total glutathione, an increase in reactive oxygen species and via a decrease in DYRK1B (dual-specificity tyrosine-regulated kinase). Nowadays, a single drug treatment of ovarian cancer is less favorable because of the increase in resistance against its cytotoxicity and using cisplatin combined with other chemotherapeutic drugs is more effective than single drug therapy to destroy the tumor by targeting different pathways such as cisplatin combined with paclitaxel in the treatment of ovarian cancer.

It has been proven that proteomic analysis can be used for drug discovery and identification of targets affected by the treatment and may open doors for a novel proteins that may play a role in the cancer metastasis or treatment [29]. To our knowledge, there is only one report that showed the proteins expressed upon cucurbitacin B treatment of breast cancer cell line (MDA-MB-231 and MCF-7) [25]. Duangmano et al. [25] studied the proteomic change by two- dimensional gel electrophoresis and were able to detect only four differentially expressed proteins. Cucurbitacin B down regulated nucleophosmin/B32 and up regulated three heat shock-related proteins (HSP70, prolyl 4-hydroxylase and chaperonin). In this article, we investigated the change in protein expression after cucurbitacin B alone or combined with cisplatin treatment for cisplatin-resistant ovarian cancer cell line (A2780CP) by using 2D-nano-UPLC-MS/MS.

Results and Discussion
Recent study in our group documented [30] the ability of cucurbitacin B to sensitize the cisplatin-resistant ovarian cancer cells (A2780CP) in both 2D and 3D culture to the cytotoxicity of cisplatin. The proteomics analysis showed that the combination treatment down regulated the protein level of Leucine-rich PPR motifcontaining protein (LRP130) (Figure 1, Supplementary material table S1). LRP130 contains 11 pentatrico peptide repeats which binds to the hydrophilic nucleotides [31]. It plays a role in the cellular homeostasis and cytoskeleton organization [23,33] through binding to mitochondrial and nuclear RNA controlling the RNA transport machinery [34]. Moreover, it has been reported that its transactivation activity in the multidrug resistance genes such as p-glycoprotein MDR1 [35] confer the antiapoptotic properties to the cancer and decrease the translocation of the drug into the cancer cells. This is the first report to demonstrate the inhibitory effect of either cucurbitacin B or cisplatin on the expression level of LRP130. This effect may lead to an increase in the amount of cisplatin in the cells and increased response of the resistant ovarian cancer cells to the cytotoxicity of cisplatin.

^aThe protein name corresponding to the entry at the Swiss Protein database.
^bFunction annotations were retrieved from Swiss Protein (http://www.uniprot.org/) and NCBInr (http://www.ncbi. nlm.nih.gov/) for human (Homo sapiens)
^cThe theoretical molecular mass or molecular weight (MW) coming from database of the protein identified.
^dThe theoretical isoelectric point (pI) coming from database of the protein identified.
^{e and f}The ratio shows the expression or relative abundance of the protein through the heat map analysis by comparing spectral counts, peptides and percentage of coverage followed by NSAF (Normalized Spectral Abundance Factor), using a “label free” approach. The values are represented by the log2 relative expression between the replicates of the biological samples of each group. Ratios ^{e and f}correspond to treated cells/control in cytoplasmic extract or nuclear extract (other cellular components except cytoplasm), respectively.
^gScores greater than 40 were taken as significant match based on MOWSE score (http://www.matrixscience.com), the threshold was set up by the server at the significance level P≥0.05 for random hit.
^hthe molecular function of each protein was retrieved from BANTHER website (http://www.pantherdb.org/).

Here in this study, the treatment of A2780CP cells with cucurbitacin B and cisplatin together significantly down regulated the level of FASN (Figure 1, Supplementary material table S1). Cucurbitacin B alone decreased the level of FASN which is a further indication of cucurbitacin B as a potential chemosensitizer for cisplatin chemotherapy (Figure 1, Supplementary material table S1). FASN plays a major role in the lipogenesis. It is considered as an oncogene in breast and prostate cancer [36,37] by stabilizing β-catenin in the cytoplasm and activation of Wnt-1/β-catenin pathway [38]. Moreover, the inhibition of fatty acid synthase activity by RNAi in osteosarcoma inhibited the cell invasion and metastasis via the inhibition of HER2/PI3K/AKT pathway [39,40]. Furthermore, its down regulation by C-75, a selective FASN inhibitor, potentiated the cytotoxicity of cisplatin in ovarian cancer via induction of apoptosis [41]. Treating the cells with cucurbitacin B and cisplatin together led to an increase in the free H2A variants in the cytoplasm (Figure 1, Supplementary material table S1) leading to genome instability and increasing the sensitivity of the cells to DNA damaging by cisplatin. The free H2Ax in the cytoplasm was found to be increased in Imatinib-treated gastrointestinal stromal tumors (GIST) a hallmark for the apoptosis induction after Erlotinib treatment [42-44].

Experimental

Ovarian Cancer Cell Lines and the Drugs
The cisplatin-sensitive (A2780) and cisplatin-resistant (A2780CP) ovarian cancer cell lines [31] were obtained from Dr. Stephen Howell, University of California, San Diego, USA. The cells were maintained in RPMI-1640 (Thermoscientific™ Hyclone™) supplemented with 10% fetal bovine serum (FBS) (Thermoscientific™ Hyclone™) and 1% penicillin (100IU/mL)/streptomycin (100μg/mL) (Corning™ Cellgro™). In order to maintain the resistance, 1μM cisplatin was added to the medium every 2-3 passages. Cisplatin (cis-diamineplatinum (II) dichloride) was obtained from Sigma and dissolved in 0.9% saline then stored as 8mM stock solution at -20°C. Cucurbitacin B was isolated from Cucurbita texana and characterized by spectroscopic techniques in our lab [46].

Cell Treatment and Fractionation for 2D-NanoLC
A2780CP cells were seeded in 25cm2 culture flask and incubated overnight. The cells were incubated either with 2μM cucurbitacin B, 40μM cisplatin for 48hrs or pre-incubated with 2μM cucurbitacin B for 24hrs then incubated for another 48hrs with 40μM cisplatin. Cell fractionation was done to increase the number of identified proteins in each sample. After cisplatin-resistant ovarian cancer cells were treated, the cells were washed with ice-cold 1x PBS and collected after trypsinzation. The cell pellet was washed with 1x PBS and re-suspended in low salt buffer (10mM HEPES pH: 7.9, 10mM KCl, 1.5mM MgCl₂.6 H₂O) and incubated 20min on ice. The cells were homogenized using 20 strokes by dounce homogenizer. The cell lysate was centrifuged at 800 × g at 4°C. The supernatant constituted a fraction enriched in cytosol components was transferred to a pre-chilled tube and lysed by 1x lysis buffer (50mM Tris-HCl pH: 7.5, 150mM NaCl, 1% Triton X-100, 50mM NaF, 1mM DTT, without protease inhibitor cocktail). The pellet enriched in the rest of the components of the cells, mainly membranes and organelles, were lysate using lysis buffer. The lysate was centrifuged at 13,000rpm for 30min, and the supernatant transferred to a new pre-chilled tube. Protein concentration was estimated by BCA protein assay kit, Pierce™.

Protein Identification by 2 Dimensional Nano-Liquid Chromatography Tandem Mass Spectrometry (2D-Nano-UPLC-MS/MS) Analysis
The proteins obtained from cell lysate (individual cytoplasmic and nuclear fractions) were in-solution reduced with DTT (Sigma Aldrich, St. Louis, MI) at 65°C for 5 minutes, followed by alkylation with Iodoacetamide (Sigma Aldrich, St. Louis, MI), and digested with sequencing grade trypsin (Promega, Madison WI) overnight at 37°C. The digested peptides were concentrated by vacuum centrifuge (Speed Vac, ThermoScientific). The trypsin digested peptides were dissolved in 100mM Ammonium Formate, pH 10, and separated through 2D-nanoLC with dilution using a 2D-nanoAcquity UPLC (Waters, Milford, MA) according to Dong G et al. [47,48].

The first dimension was performed in XBridge BEH130 C18, 5μm, 300μm×50mm NanoEase Column (Waters, Milford, MA) using solvent A1: 20mM Ammonium Formate, pH=10 and B1: 100% Acetonitrile (Fisher Optima, LC-MS grade). The flow at 1st dimension was 2μL/min and 10 different step gradients (dilution method) were performed for 20 minute separately. The second dimension included trapping and desalting online through 180μm×20mm, 5μm Symmetry C18 nanoAcquity UPLC trap column (Waters, Milford, MA) at flow = 20μL/min, 99% A2 (H2O, 0.1% Formic Acid) and 1% B2 (100% Acetonitrile, 0.1% Formic Acid) for 20 minutes. After the peptides were desalted and concentrated, they were separated online in the second dimension through BEH130 C18 1.7um, 100 μm×100 mm nanoAcquity UPLC column (Waters, Milford, MA). The standard solvent gradient used was: 0-2min, 3% B2 isocratic; 2-40min, 3-85% B2 linear, at a flow rate of 400nL/min for 60 minutes. The eluted ions were analyzed by one full precursor MS scan (400-150m/z) followed by four MS/MS scans of the most abundant ions detected in the precursor MS scan while operating under dynamic exclusion or direct data acquisition system (Table 1). Spectra obtained in the positive ion mode with nano ESI-Q-Tof Synapt G1 mass spectrometer (Waters, Milford, MA) were deconvoluted, and analyzed using the MassLynx software 4.1 (Micromass, UK), and exported to ProteinLynx Global Server v3.0 (PLGS v3.0) (Waters, Milford, MA) to creates the list of masses. A peak list (PKL format) was generated to identify +1 or multiple charged precursor ions from the mass spectrometry data file (Table S2). The instrument was calibrated in MS/MS mode using 100 fmole of (Glu1) - Fibrinopeptide B human (Sigma, Saint Louis, MO) with a RMS residual of 3.857 e-4 amu or 6.9413 e-1 ppm. Parent mass (MS) and fragment mass (MS/MS) peak ranges were 400-2000 Da and 65- 2000 Da, respectively.

Database Searching
Mascot server v2.5.0 and Mascot Daemon Toolbox v2.5.0 (www.matrix-science.com, UK) in MS/MS ion search mode (local licenses) were applied to conduct peptide matches (peptide masses and sequence tags) and protein searches against Swiss Protein 2015_03 (547964 sequences; 195174196 residues) using taxonomy filter Homo sapiens (humans) (20203 sequences). The following parameters were set for the search: carbamidomethyl (C) on cysteine was set as fixed; variable modifications included asparagine and glutamine deamination and methionine oxidation, as well as Error Tolerance mode. One missed cleavage was allowed for regular searching; monoisotopic masses were counted; the precursor peptide mass tolerance was set at 50ppm; fragment mass tolerance was 0.3 Da and the ion score or expected cut-off was set at 5. The MS/MS spectra were searched with MASCOT using a 95% confidence interval (C.I. %) threshold (p < 0.05), with which minimum score of 40 was used for peptide identification. All proteins identified were found these domains. Also, they were corroborated with X! Tandem (GPM) (www.thegpm.org), an open source setting using similar setup like Mascot (enzyme, cleavages, and modifications) and ENSEMBL Homo sapiens database (http://ftp.ensembl.org/pub/release-79/embl/homo_sapiens/).

The protein relative abundance analysis was performed using a “label free” approach through the measure of the spectral counts and the ratio reported as the log2 relative expression. ProteoIQ software v2.7 (www. premierbiosoft.com) was used for the post-mass spectrometry bioinformatics analysis (ratio calculation and normalization by NSAF=Normalized Spectral Abundance Factor, as well as the log2 relative expression) mentioned before. The proteins accepted from the identification list met the following criteria: >99.0% probability to achieve a false discovery rate (FDR) of < 1% [49,50] (the analysis was performed using SP with taxonomy filter for human target and decoy databases at ProteoIQ v2.7). Moreover, the proteins identified were corroborate by the observation of the False Positive Rate (FPR) coming from the analysis by GPM as second approach (the FPR average was 0.62%) [51,52].

Gene Ontology Annotation
The differentially expressed proteins were submitted to PANTHER database to identify the molecular function, biological process and pathways of the proteins (http://www.pantherdb.org/). Proteins that are involved in the survival of cancer patients were identified by using Bioprofiling website (http://www. bioprofiling.de/GEO/GENESURV/genesurv.html). GPM server was used to identify the enrichment and depleted proteins in the protein list.

Western Blot Confirmation for Proteomic Results
The cisplatin-resistant ovarian cancer cells were seeded in 60 mm culture dish (5*104cells/mL, 3mL/dish) and allowed to adhere overnight at 37°C and 5% CO2. The cells were incubated either with 2μM cucurbitacin B, 40μM cisplatin for 48hrs or pre-incubated with 2μM cucurbitacin B for 24hrs then further incubated for another 48hrs with 40μM cisplatin. The cells were washed 2 times with 1x PBS and lysed by 1x RIPA buffer containing 1x protease and phosphatase inhibitor cocktail. The protein content was quantified by BCA protein assay kit. 20 or 40μg/well was separated on SDS-PAGE.

The protein was transferred to a nitrocellulose membrane 0.2 or 0.45μm for 45 or 90minutes at 100 or 90 V, respectively. The membrane was reacted with the primary antibodies (Table S3). β-actin was used as a loading control. The secondary antibody (Table S3) was applied to the membrane and was detected either with the GE Healthcare Amersham ECL Plus Western Blotting Detection Reagents (Piscataway, NJ) and the signal was recorded by a UVP gel documentation system (UVP, Upland, CA) or by Odyssey® Imager. The level of protein expression was normalized to the level of the loading control β-actin then the fold of change was determined by comparing the band intensity of the control cells to the untreated cells. Statistical analysis was done using the software SPSS 19 for Microsoft Windows (SPSS Inc, Chicago, Illinois). The fold of change in Fatty acid synthase, LRP-130 and Histone H2A.X level was tested using one-way analysis of variance (ANOVA). The fold of change was considered significant when (P < 0.05).

Conclusion

Overcoming the ovarian cancer resistance will led to a better survival rate of the patients. Using natural products such as cucurbitacin B at low concentration with the common chemotherapeutic drug, will improve the cytotoxicity of the drug with minimal side effects. In our report, cucurbitacin B alone was able to alter the expression of vital proteins required to regulate different pathways in the tumor cells. When cucurbitacin B combined with cisplatin, they efficiently kill the tumor cells by changing the expression of wide range of proteins that control survival signaling pathways.

Here we show that this combination worth further investigation on the level of in vivo studies followed by a clinical trial.

Supplementary Files
The procedures for all assays including cell treatment and fractionation for 2D-nanoLC, protein identification by 2 dimensional nano-liquid chromatography tandem mass spectrometry (2D-nano-UPLC-MS/MS) analysis, database searching, gene ontology annotation and western blot confirmation for proteomic results are reported in the supplementary file.

Acknowledgements
This work was supported by the Egyptian government via the Egyptian Ministry of Higher Education and Scientific Research and was supported by the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20GM103443 and P20RR016479.

Bibliography

1. Siegel, R. L., Miller, K. D. & Jemal, A. (2015). Cancer statistics, 2015. CA: a cancer journal for clinicians, 65(1), 5-29.
2. Siegel, R. L., Miller, K. D. & Jemal, A. (2016). Cancer statistics, 2016. CA: a cancer journal for clinicians, 66(1), 7-30.
3. Galluzzi, L., Senovilla, L., Vitale, I., Michels, J., Martins, I., Kepp, O., Castedo, M. & Kroemer, G. (2012). Molecular mechanisms of cisplatin resistance. Oncogene., 31(15), 1869-1883.
4. Alghasham, A. A. (2013). Cucurbitacins - a promising target for cancer therapy. Int J Health Sci (Qassim)., 7(1), 77-89.
5. Chen, J. C., Chiu, M. H., Nie, R. L., Cordell, G. A. & Qiu, S. X. (2005). Cucurbitacins and cucurbitane glycosides: structures and biological activities. Nat Prod Rep., 22(3), 386-399.
6. Chen, X., Bao, J., Guo, J., Ding, Q., Lu, J., Huang, M. & Wang, Y. (2012). Biological activities and potential molecular targets of cucurbitacins: a focus on cancer. Anticancer Drugs, 23(8), 777-787.
7. Chan, K. T., Li, K., Liu, S. L., Chu, K. H., Toh, M. & Xie, W. D. (2010). Cucurbitacin B inhibits STAT3 and the Raf/MEK/ERK pathway in leukemia cell line K562. Cancer Lett., 289(1), 46-52.
8. Chan, K. T., Meng, F. Y., Li, Q., Ho, C. Y., Lam, T. S., To, Y., Lee, W. H., Li, M., Chu, K. H. & Toh, M. (2010). Cucurbitacin B induces apoptosis and S phase cell cycle arrest in BEL-7402 human hepatocellular carcinoma cells and is effective via oral administration. Cancer Lett., 294(1), 118-124.
9. El-Senduny, F. F., Badria, F. A., El-waseef, A. M., Chauhan, S. C. & Halaweish, F (2016). Approach for chemosensitization of cisplatin-resistant ovarian cancer by cucurbitacin B. Tumor Biology, 37(1), 685-698.
10. Haritunians, T., Gueller, S., Zhang, L., Badr, R., Yin, D., Xing, H., Fung, M. C. & Koeffler, H. P. (2008). Cucurbitacin B induces differentiation, cell cycle arrest, and actin cytoskeletal alterations in myeloid leukemia cells. Leuk Res., 32(9), 1366-1373.
11. Liu, T., Zhang, M., Deng, Y., Zhang, H., Sun, C., Yang, X. & Ji, W. (2008). [Effects of cucurbitacin B on cell proliferation and apoptosis in Hep-2 cells]. Lin Chung Er Bi Yan Hou Tou Jing Wai Ke Za Zhi., 22(9), 403-407.
12. Lu, P., Yu, B. & Xu, J. (2012). Cucurbitacin B regulates immature myeloid cell differentiation and enhances antitumor immunity in patients with lung cancer. Cancer Biother Radiopharm, 27(8), 495-503.
13. Promkan, M., Dakeng, S., Chakrabarty, S., Bogler, O. & Patmasiriwat, P. (2013). The effectiveness of cucurbitacin B in BRCA1 defective breast cancer cells. PLoS One., 8(2).
14. Wakimoto, N., Yin, D., O'Kelly, J., Haritunians, T., Karlan, B., Said, J., Xing, H. & Koeffler, H. P. (2008). Cucurbitacin B has a potent antiproliferative effect on breast cancer cells in vitro and in vivo. Cancer Sci., 99(9), 1793-1797.
15. Yasuda, S., Yogosawa, S., Izutani, Y., Nakamura, Y., Watanabe, H. & Sakai, T. (2010). Cucurbitacin B induces G2 arrest and apoptosis via a reactive oxygen species-dependent mechanism in human colon adenocarcinoma SW480 cells. Mol Nutr Food Res., 54(4), 559-565.
16. Yin, D., Wakimoto, N., Xing, H., Lu, D., Huynh, T., Wang, X., Black, K. L. & Koeffler, H. P. (2008). Cucurbitacin B markedly inhibits growth and rapidly affects the cytoskeleton in glioblastoma multiforme. Int J Cancer., 123(6), 1364-1375.
17. Zhang, M., Sun, C., Shan, X.., Yang, X., Li-Ling, J. & Deng, Y. (2010). Inhibition of pancreatic cancer cell growth by cucurbitacin B through modulation of signal transducer and activator of transcription 3 signaling. Pancreas, 39(6), 923-929.
18. Lee, D. H., Thoennissen, N. H., Goff, C., Iwanski, G. B., Forscher, C., Doan, N. B., Said, J. W. & Koeffler, H. P. (2011). Synergistic effect of low-dose cucurbitacin B and low-dose methotrexate for treatment of human osteosarcoma. Cancer Lett., 306(2), 161-170.
19. Chen, W., Leiter, A., Yin, D., Meiring, M., Louw, V. J. & Koeffler, H. P. (2010). Cucurbitacin B inhibits growth, arrests the cell cycle, and potentiates antiproliferative efficacy of cisplatin in cutaneous squamous cell carcinoma cell lines. Int J Oncol., 37(3), 737-743.
20. Liu, T., Peng, H., Zhang, M., Deng, Y. & Wu, Z. (2010). Cucurbitacin B, a small molecule inhibitor of the Stat3 signaling pathway, enhances the chemosensitivity of laryngeal squamous cell carcinoma cells to cisplatin. Eur J Pharmacol., 641(1), 15-22.
21. Iwanski, G. B., Lee, D. H., En-Gal, S., Doan, N. B., Castor, B., Vogt, M., Toh, M., et al. (2010). Cucurbitacin B, a novel in vivo potentiator of gemcitabine with low toxicity in the treatment of pancreatic cancer. Br J Pharmacol., 160(4), 998-1007.
22. Thoennissen, N. H., Iwanski, G. B., Doan, N. B., Okamoto, R., Lin, P., Abbassi, S., Song, J. H., Yin, D., Toh, M., Xie, W. D., Said, J. W. & Koeffler, H. P. (2009). Cucurbitacin B induces apoptosis by inhibition of the JAK/STAT pathway and potentiates antiproliferative effects of gemcitabine on pancreatic cancer cells. Cancer Res., 69(14), 5876-5884.
23. Liu, T., Zhang, M., Zhang, H., Sun, C., Yang, X., Deng, Y. & Ji, W. (2008). Combined antitumor activity of cucurbitacin B and docetaxel in laryngeal cancer. Eur J Pharmacol., 587(1-3), 78-84.
24. Duangmano, S., Sae-Lim, P., Suksamrarn. A., Patmasiriwat, P & Domann, E. F. (2012). Cucurbitacin B Causes Increased Radiation Sensitivity of Human Breast Cancer Cells via G2/M Cell Cycle Arrest. J Oncol., 601682.
25. Duangmano, S., Sae-Lim, P., Suksamrarn, A., Domann, F. E. & Patmasiriwat, P (2012). Cucurbitacin B inhibits human breast cancer cell proliferation through disruption of microtubule polymerization and nucleophosmin/B23 translocation. BMC Complement Altern Med., 12, 185.
26. Zhu, J. S., Ouyang, D. Y., Shi, Z. J., Xu, L. H., Zhang, Y. T. & He, X. H. (2012). Cucurbitacin B induces cell cycle arrest, apoptosis and autophagy associated with G actin reduction and persistent activation of cofilin in Jurkat cells. Pharmacology, 89(5-6), 348-346.
27. Wiart, C. (2013). The definition and significance of Cucurbitacin B a STAT3 inhibitors. Cancer Lett., 328(1), 188.
28. Kausar, H., Munagala, R., Bansal, S. S., Aqil, F., Vadhanam, M. V. & Gupta, R. C. (2013). Cucurbitacin B potently suppresses non-small-cell lung cancer growth: identification of intracellular thiols as critical targets. Cancer Lett., 332(1), 35-45.
29. Burbaum, J. & Tobal, G. M. (2002). Proteomics in drug discovery. Curr Opin Chem Biol., 6(4), 427-433.
30. El-Senduny, F. F., Badria, F. A., El-Waseef, A. M., Chauhan, S. C. & Halaweish, F. (2015). Approach for chemosensitization of cisplatin-resistant ovarian cancer by cucurbitacin B. Tumour Biol., 37(1), 3773-3778.
31. Hou, J., Wang, F. & McKeehan, W. L. (1994). Molecular cloning and expression of the gene for a major leucine-rich protein from human hepatoblastoma cells (HepG2). In Vitro Cell Dev Biol Anim., 30A(2), 111-114.
32. Liu, L. & McKeehan, W. L. (2002). Sequence analysis of LRPPRC and its SEC1 domain interaction partners suggests roles in cytoskeletal organization, vesicular trafficking, nucleocytosolic shuttling, and chromosome activity. Genomics, 79(1), 124-136.
33. Mootha, V. K., Lepage, P., Miller, K., Bunkenborg, J., Reich, M., Hjerrild, M., Delmonte, T., Villeneuve, A., et al. (2003). Identification of a gene causing human cytochrome c oxidase deficiency by integrative genomics. Proc Natl Acad Sci U S A., 100(2), 605-610.
34. Mili, S. & Pinol-Roma, S. (2003). LRP130, a pentatricopeptide motif protein with a noncanonical RNA-binding domain, is bound in vivo to mitochondrial and nuclear RNAs. Mol Cell Biol., 23(14), 4972-4982.
35. Michaud, M., Barakat, S., Magnard, S., Rigal, D. & Baggetto, L. G. (2011). Leucine-rich protein 130 contributes to apoptosis resistance of human hepatocarcinoma cells. Int J Oncol., 38(1), 169-178.
36. Migita, T., Ruiz, S., Fornari, A., Fiorentino, M., Priolo, C., Zadra, G., et al. (2009). Fatty acid synthase: a metabolic enzyme and candidate oncogene in prostate cancer. Journal of the National Cancer Institute, 101(7), 519-532.
37. Vazque-Martin, A., Colomer, R., Brunet, J., Lupu, R. & Menendez, J. (2008). Overexpression of fatty acid synthase gene activates HER1/HER2 tyrosine kinase receptors in human breast epithelial cells. Cell Proliferation, 41(1), 59-85.
38. Fiorentino, M., Zadra, G., Palescandolo, E., Fedele, G., Bailey, D., Fiore, C., et al. (2008). Overexpression of fatty acid synthase is associated with palmitoylation of Wnt1 and cytoplasmic stabilization of β-catenin in prostate cancer. Laboratory investigation, 88(12), 1340-1348.
39. Liu, Z. L., Mao, J. H., Peng, A. F., Yin, Q. S., Zhou, Y., Long, X. H. & S. H. Huang. (2013). Inhibition of fatty acid synthase suppresses osteosarcoma cell invasion and migration via downregulation of the PI3K/Akt signaling pathway in vitro. Mol Med Rep., 7(2), 608-612.
40. Wang, T. F., Wang, H., Peng, A. F., Luo, Q. F., Liu, Z. L., Zhou, R. P., Gao, S., Zhou, Y. & Chen, W. Z. (2013). Inhibition of fatty acid synthase suppresses U-2 OS cell invasion and migration via downregulating the activity of HER2/PI3K/AKT signaling pathway in vitro. Biochem Biophys Res Commun., 440(2), 229-234.
41. Uddin, S., Jehan, Z., Ahmed, M., Alyan, A., Al-Dayel, F., Hussain, A., Bavi, P. & Al-Kuraya, K. S. (2011). Overexpression of fatty acid synthase in Middle Eastern epithelial ovarian carcinoma activates AKT and Its inhibition potentiates cisplatin-induced apoptosis. Mol Med., 17(7-8), 635-645.
42. Liu, Y., Tseng, M., Perdreau, S. A., Rossi, F., Antonescu, C., Besmer, P., et al. (2007). Histone H2AX Is a Mediator of Gastrointestinal Stromal Tumor Cell Apoptosis following Treatment with Imatinib Mesylate. Cancer Research, 67(6), 2685-2692.
43. Monteiro, F. L., Baptista, T., Amado, F., Vitorino, R., Jeronimo, C. & Helguero, L. A. (2014). Expression and functionality of histone H2A variants in cancer. Oncotarget, 5(11), 3428-3443.
44. Liu, Y., Tseng, M., Perdreau, S. A., Rossi, F., Antonescu, C., Besmer, P., Fletcher, J. A., Duensing, S. & Duensing, A. (2007). Histone H2AX Is a Mediator of Gastrointestinal Stromal Tumor Cell Apoptosis following Treatment with Imatinib Mesylate. Cancer Research, 67(6), 2685-2692.
45. Hamaguchi, K., Godwin, A. K., Yakushiji, M., O'Dwyer, P. J., Ozols, R. F. & Hamilton, T. C. (1993). Cross-Resistance to Diverse Drugs Is Associated with Primary Cisplatin Resistance in Ovarian Cancer Cell Lines. Cancer Research, 53(21), 5225-5232.
46. Bartalis, J. & Halaweish, F. T. (2011). In vitro and QSAR studies of cucurbitacins on HepG2 and HSC-T6 liver cell lines. Bioorg Med Chem., 19(8), 2757-2766.
47. Dong, G., Callegari, E., Gloeckner, C. J., Ueffing, M. & Wang, H. (2012). Mass spectrometric identification of novel posttranslational modification sites in Huntingtin. Proteomics, 12(12), 2060-2064.
48. Dong, G., Callegari, E. A., Gloeckner, C. J., Ueffing, M. & Wang, H. (2012). Prothymosin-alpha interacts with mutant huntingtin and suppresses its cytotoxicity in cell culture. J Biol Chem., 287(2), 1279-1289.
49. Keller, A., Nesvizhskii, A. I., Kolker, E. & Aebersold, R. (2002). Empirical statistical model to estimate the accuracy of peptide identifications made by MS/MS and database search. Analytical Chemistry, 74(20), 5383-5392.
50. Nesvizhskii, A. I., Keller, A., Kolker, E. & Aebersold, R. (2003). A statistical model for identifying proteins by tandem mass spectrometry. Analytical chemistry, 75(17), 4646-4658.
51. Gupta, N., Bandeira, N., Keich, U. & Pevzner, P. A. (2011). Target-decoy approach and false discovery rate: when things may go wrong. Journal of the American Society for Mass Spectrometry, 22(7), 1111-1120.
52. Ghiso, N. S. & Lennon, G. G. (1994). lrp130 gene assigned to chromosome 2. In Vitro Cell Dev Biol Anim., 30A(11), 744.