Ramona Britto

Publications of Ramona Britto

• Cytotoxicity of half sandwich ruthenium(II) complexes with strong hydrogen bond acceptor ligands and their mechanism of action.

  Authors: Sangeeta Das, Sarika Sinha, Ramona Britto, Kumaravel Somasundaram, Ashoka G Samuelson

  Journal of inorganic biochemistry. 10/2009;

  Neutral and cationic organometallic ruthenium(II) piano stool complexes of the type [(eta(6)-cymene)RuCl(X)(Y)] (complexes R1-R8) has been synthesized and characterized. In cationic complexes, X, YNeutral and cationic organometallic ruthenium(II) piano stool complexes of the type [(eta(6)-cymene)RuCl(X)(Y)] (complexes R1-R8) has been synthesized and characterized. In cationic complexes, X, Y is either a eta(2) phosphorus ligand such as 1,1-bis(diphenylphosphino)methane (DPPM) and 1,2-bis(diphenylphosphino)ethane (DPPE) or partially oxidized ligands such as 1,2-bis(diphenylphosphino)methane monooxide (DPPMO) and 1,2-bis(diphenylphosphino)ethane monooxide (DPPEO) which are strong hydrogen bond acceptors. In neutral complexes, X is chloride and Y is a monodentate phosphorous donor. Complexes with DPPM and DPPMO ligands ([(eta(6)-cymene)Ru(eta(2)-DPPM)Cl]PF(6) (R2), [(eta(6)-cymene)Ru(eta(2)-DPPMO)Cl]PF(6) (R3), [(eta(6)-cymene)Ru(eta(1)-DPPM)Cl(2)] (R5) and [(eta(6)-cymene)Ru(eta(1)-DPPMO)Cl(2)] (R6) show good cytotoxicity. Growth inhibition study of several human cancer cell lines by these complexes has been carried out. Mechanistic studies for R5 and R6 show that inhibition of cancer cell growth involves both cell cycle arrest and apoptosis induction. Using an apoptosis PCR array, we identified the sets of anti-apoptotic genes that were down regulated and pro-apoptotic genes that were up regulated. These complexes were also found to be potent metastasis inhibitors as they prevented cell invasion through matrigel. The complexes were shown to bind DNA in a non intercalative fashion and cause unwinding of plasmid DNA in cell-free medium by competitive ethidium bromide binding, viscosity measurements, thermal denaturation and gel mobility shift assays.

• Novel glioblastoma markers with diagnostic and prognostic value identified through transcriptome analysis.

  Authors: Sreekanth P Reddy, Ramona Britto, Katyayni Vinnakota, Hebbar Aparna, Hari Kishore Sreepathi, Balaram Thota, Arpana Kumari, B M Shilpa, M Vrinda, Srikantha Umesh [......] Ashwani Tandon, Paritosh Pandey, Sridevi Hegde, A S Hegde, Anandh Balasubramaniam, B A Chandramouli, Vani Santosh, Paturu Kondaiah, Kumaravel Somasundaram, M R Satyanarayana Rao

  Clinical cancer research : an official journal of the American Association for Cancer Research. 06/2008; 14(10):2978-87.

  PURPOSE: Current methods of classification of astrocytoma based on histopathologic methods are often subjective and less accurate. Although patients with glioblastoma have grave prognosis,PURPOSE: Current methods of classification of astrocytoma based on histopathologic methods are often subjective and less accurate. Although patients with glioblastoma have grave prognosis, significant variability in patient outcome is observed. Therefore, the aim of this study was to identify glioblastoma diagnostic and prognostic markers through microarray analysis. EXPERIMENTAL DESIGN: We carried out transcriptome analysis of 25 diffusely infiltrating astrocytoma samples [WHO grade II--diffuse astrocytoma, grade III--anaplastic astrocytoma, and grade IV--glioblastoma (GBM)] using cDNA microarrays containing 18,981 genes. Several of the markers identified were also validated by real-time reverse transcription quantitative PCR and immunohistochemical analysis on an independent set of tumor samples (n = 100). Survival analysis was carried out for two markers on another independent set of retrospective cases (n = 51). RESULTS: We identified several differentially regulated grade-specific genes. Independent validation by real-time reverse transcription quantitative PCR analysis found growth arrest and DNA-damage-inducible alpha (GADD45alpha) and follistatin-like 1 (FSTL1) to be up-regulated in most GBMs (both primary and secondary), whereas superoxide dismutase 2 and adipocyte enhancer binding protein 1 were up-regulated in the majority of primary GBM. Further, identification of the grade-specific expression of GADD45alpha and FSTL1 by immunohistochemical staining reinforced our findings. Analysis of retrospective GBM cases with known survival data revealed that cytoplasmic overexpression of GADD45alpha conferred better survival while the coexpression of FSTL1 with p53 was associated with poor survival. CONCLUSIONS: Our study reveals that GADD45alpha and FSTLI are GBM-specific whereas superoxide dismutase 2 and adipocyte enhancer binding protein 1 are primary GBM-specific diagnostic markers. Whereas GADD45alpha overexpression confers a favorable prognosis, FSTL1 overexpression is a hallmark of poor prognosis in GBM patients.

• Shift in AP-2alpha localization characterizes astrocytoma progression.

  Authors: Ramona Britto, S Umesh, A S Hegde, Sridevi Hegde, Vani Santosh, B A Chandramouli, Kumaravel Somasundaram

  Cancer biology & therapy. 04/2007; 6(3):413-8.

  Activator protein 2alpha (AP-2alpha) has been shown to be lost in the advanced stages of many cancers, including gliomas. In this study, we wanted to analyze the expression of AP-2alpha inActivator protein 2alpha (AP-2alpha) has been shown to be lost in the advanced stages of many cancers, including gliomas. In this study, we wanted to analyze the expression of AP-2alpha in astrocytoma samples of different grades both at the RNA level, by real-time qPCR and at the protein level, by immunohistochemistry, and to examine its correlation, if any, with patient outcome. Five Grade I, 14 Grade II, 18 Grade III, 72 Grade IV samples and 13 normal brain controls were included. We did not find any clear pattern of regulation at the RNA level with tumor grade. The RNA expression levels however, correlated to a large extent with the nuclear AP-2alpha staining in these samples (72.09%; 31/43). Further, we did not find a complete loss of nuclear AP-2alpha expression in the higher grades, in contrast to previous reports. Interestingly, we found cytoplasmic AP-2alpha expression in a majority of higher grade astrocytomas (Grade IV-85%; 33/39 and Grade III-74%; 14/19) in comparison to lower grades (Grade I-0%; 0/5 and Grade II-37.5%; 3/8) suggesting that the translocation of this protein from the nucleus to the cytoplasm may be responsible for the increased malignancy. The nuclear expression in these grades was found to be concomitantly reduced. Within GBMs, we found that decreased nuclear expression was indicative of a better prognosis. The striking observation was the shift in localization of this protein from the nucleus to the cytoplasm with increasing tumor grade, pointing to a crucial role for this transcription factor in the progression of astrocytomas.

• Apoptosis induction by activator protein 2alpha involves transcriptional repression of Bcl-2.

  Authors: Narendra Wajapeyee, Ramona Britto, Halasahalli M Ravishankar, Kumaravel Somasundaram

  The Journal of biological chemistry. 07/2006; 281(24):16207-19.

  Activator protein 2alpha (AP-2alpha) induces cytotoxicity by inducing cell cycle arrest and apoptosis. In this study we investigated the mechanism of apoptosis induction by AP-2alpha. We found thatActivator protein 2alpha (AP-2alpha) induces cytotoxicity by inducing cell cycle arrest and apoptosis. In this study we investigated the mechanism of apoptosis induction by AP-2alpha. We found that AP-2alpha induced apoptosis efficiently in cells treated with benzyloxycar-bonyl-IETD-fluoromethyl ketone or FADD-silenced cells but failed to do so in benzyloxycarbonyl-LEHD-fluoromethyl ketone-treated or apoptosis protease activation factor-1 (Apaf1)-silenced cells, suggesting the central role of mitochondria in AP-2alpha-induced apoptosis. In good correlation, cells overexpressing AP-2alpha showed a reduction in mitochondrial membrane potential (Deltapsi(m)), cytochrome c and Smac/DIABLO release into cytosol, and Bax translocation into mitochondria. We found that the pro-apoptotic protein Bax is important for AP-2alpha-induced apoptosis as adenovirus AP2 failed to induce apoptosis in HCT116 Bax(-/-) cells. However, we found the IAP (inhibitor of apoptosis) inhibitor Smac/DIABLO may have a limited role in AP-2alpha-induced apoptosis as we found the IAP member Survivin down-regulated by AP-2alpha. Although the total Bax level remains unaltered, we found a time-dependent increase in the activated form of Bax in adenovirus AP2-infected cells. In addition, we show that AP-2alpha transcriptionally represses Bcl-2 by binding to its promoter both in vitro and in vivo and that this is essential for AP-2alpha-induced apoptosis as ectopic expression of Bcl-2 efficiently inhibited apoptosis induced by AP-2alpha. Furthermore, we show that chemotherapy-induced endogenous AP-2alpha down-regulates Bcl-2 and induces apoptosis in an AP-2alpha-dependent manner. Moreover, we demonstrate that inhibition of okadaic acid or staurosporine-sensitive pathways in AP-2alpha overexpressing breast cancer cells resulted in AP-2alpha-dependent apoptosis induction. These results suggest that AP-2alpha induces apoptosis by down-regulating Bcl-2 and utilizing a bax/cytochrome c/Apaf1/caspase 9-dependent mitochondrial pathway.

• Upregulation of ASCL1 and inhibition of Notch signaling pathway characterize progressive astrocytoma.

  Authors: Kumaravel Somasundaram, Sreekanth P Reddy, Katyayni Vinnakota, Ramona Britto, Madhavan Subbarayan, Sandeep Nambiar, Aparna Hebbar, Cini Samuel, Mitesh Shetty, Hari Kishore Sreepathi, Vani Santosh, Alangar Sathyaranjandas Hegde, Sridevi Hegde, Paturu Kondaiah, M R S Rao

  Oncogene. 11/2005; 24(47):7073-83.

  Astrocytoma is the most common type of brain cancer constituting more than half of all brain tumors. With an aim to identify markers describing astrocytoma progression, we have carried out microarrayAstrocytoma is the most common type of brain cancer constituting more than half of all brain tumors. With an aim to identify markers describing astrocytoma progression, we have carried out microarray analysis of astrocytoma samples of different grades using cDNA microarray containing 1152 cancer-specific genes. Data analysis identified several differentially regulated genes between normal brain tissue and astrocytoma as well as between grades II/III astrocytoma and glioblastoma multiforme (GBM; grade IV). We found several genes known to be involved in malignancy including Achaete-scute complex-like 1 (Drosophila) (ASCL1; Hash 1). As ASCL has been implicated in neuroendocrine, medullary thyroid and small-cell lung cancers, we chose to examine the role of ASCL1 in the astrocytoma development. Our data revealed that ASCL1 is overexpressed in progressive astrocytoma as evidenced by increased levels of ASCL1 transcripts in 85.71% (6/7) of grade II diffuse astrocytoma (DA), 90% (9/10) of grade III anaplastic astrocytoma (AA) and 87.5% (7/8) of secondary GBMs, while the majority of primary de novo GBMs expressed similar to or less than normal brain levels (66.67%; 8/12). ASCL1 upregulation in progressive astrocytoma is accompanied by inhibition of Notch signaling as seen by uninduced levels of HES1, a transcriptional target of Notch1, increased levels of HES6, a dominant-negative inhibitor of HES1-mediated repression of ASCL1, and increased levels of Notch ligand Delta1, which is capable of inhibiting Notch signaling by forming intracellular Notch ligand autonomous complexes. Our results imply that inhibition of Notch signaling may be an important early event in the development of grade II DA and subsequent progression to grade III AA and secondary GBM. Furthermore, ASCL1 appears to be a putative marker to distinguish primary GBM from secondary GBM.

• Ribozyme's potential in cervical cancer control.

  Authors: Ramona Britto, Kumaravel Somasundaram

  Cancer biology & therapy. 12/2004; 3(11):1135-6.

• Upregulation of ASCL1 and inhibition of Notch signaling pathway characterize progressive astrocytoma

  Authors: Kumaravel Somasundaram, Sreekanth P Reddy, Katyayni Vinnakota, Ramona Britto, Madhavan Subbarayan, Sandeep Nambiar, Aparna Hebbar, Cini Samuel, Mitesh Shetty, Hari Kishore Sreepathi, Vani Santosh, Alangar Sathyaranjandas Hegde, Sridevi Hegde, Paturu Kondaiah, MRS Rao

  Astrocytoma is the most common type of brain cancer constituting more than half of all brain tumors. With an aim to identify markers describing astrocytoma progression, we have carried out microarrayAstrocytoma is the most common type of brain cancer constituting more than half of all brain tumors. With an aim to identify markers describing astrocytoma progression, we have carried out microarray analysis of astrocytoma samples of different grades using cDNA microarray containing 1152 cancer-specific genes. Data analysis identified several differentially regulated genes between normal brain tissue and astrocytoma as well as between grades II/III astrocytoma and glioblastoma multiforme (GBM; grade IV). We found several genes known to be involved in malignancy including Achaete-scute complex-like1(Drosophila) (ASCL1; Hash 1). As ASCL has been implicated in neuroendocrine, medullary thyroid and small-cell lung cancers, we chose to examine the role of ASCL1 in the astrocytoma development. Our data revealed that ASCL1 is over expressed in progressive astrocytoma as evidenced by increased levels of ASCL1 transcripts in 85.71% (6/7) of grade II diffuse astrocytoma (DA), 90% (9/10) of grade III anaplastic astrocytoma (AA) and 87.5% (7/8) of secondary GBMs, while the majority of primary de novo GBMs expressed similar to or less than normal brain levels (66.67%; 8/12). ASCL1 up regulation in progressive astrocytoma is accompanied by inhibition of Notch signaling as seen by uninduced levels of HES1, a transcriptional target of Notch1, increased levels of HES6, a dominant-negative inhibitor of HES1-mediated repression of ASCL1, and increased levels of Notch ligand Delta1, which is capable of inhibiting Notch signaling by forming intracellular Notch ligand autonomous complexes. Our results imply that inhibition of Notch signaling may be an important early event in the development of grade II DA and subsequent progression to grade III AA and secondary GBM.Furthermore, ASCL1 appears to be a putative marker to distinguish primary GBM from secondary GBM.

• Cytotoxicity of half sandwich ruthenium(II) complexes with strong hydrogen bond acceptor ligands and their mechanism of action

  Authors: Sangeeta Das, Sarika Sinha, Ramona Britto, Kumaravel Somasundaram, Ashoka G. Samuelson

  Journal of Inorganic Biochemistry.

  Neutral and cationic organometallic ruthenium(II) piano stool complexes of the type [(η6-cymene)RuCl(X)(Y)] (complexes R1–R8) has been synthesized and characterized. In cationic complexes, X, Y isNeutral and cationic organometallic ruthenium(II) piano stool complexes of the type [(η6-cymene)RuCl(X)(Y)] (complexes R1–R8) has been synthesized and characterized. In cationic complexes, X, Y is either a η2 phosphorus ligand such as 1,1-bis(diphenylphosphino)methane (DPPM) and 1,2-bis(diphenylphosphino)ethane (DPPE) or partially oxidized ligands such as 1,2-bis(diphenylphosphino)methane monooxide (DPPMO) and 1,2-bis(diphenylphosphino)ethane monooxide (DPPEO) which are strong hydrogen bond acceptors. In neutral complexes, X is chloride and Y is a monodentate phosphorous donor. Complexes with DPPM and DPPMO ligands ([(η6-cymene)Ru(η2-DPPM)Cl]PF6 (R2), [(η6-cymene)Ru(η2-DPPMO)Cl]PF6 (R3), [(η6-cymene)Ru(η1-DPPM)Cl2] (R5) and [(η6-cymene)Ru(η1-DPPMO)Cl2] (R6) show good cytotoxicity. Growth inhibition study of several human cancer cell lines by these complexes has been carried out. Mechanistic studies for R5 and R6 show that inhibition of cancer cell growth involves both cell cycle arrest and apoptosis induction. Using an apoptosis PCR array, we identified the sets of anti-apoptotic genes that were down regulated and pro-apoptotic genes that were up regulated. These complexes were also found to be potent metastasis inhibitors as they prevented cell invasion through matrigel. The complexes were shown to bind DNA in a non intercalative fashion and cause unwinding of plasmid DNA in cell-free medium by competitive ethidium bromide binding, viscosity measurements, thermal denaturation and gel mobility shift assays.

• Shift in $AP-2\alpha$ Localization Characterizes Astrocytoma Progression

  Authors: Ramona Britto, S Umesh, AS Hegde, Sridevi Hegde, Vani Santosh, BA Chandramouli, Kumaravel Somasundaram

  Activator protein $2\alpha$ $(AP‑2\alpha)$ has been shown to be lost in the advanced stages of many cancers, including gliomas. In this study, we wanted to analyze the expression of $(AP‑2\alpha)$ inActivator protein $2\alpha$ $(AP‑2\alpha)$ has been shown to be lost in the advanced stages of many cancers, including gliomas. In this study, we wanted to analyze the expression of $(AP‑2\alpha)$ in astrocytoma samples of different grades both at the RNA level, by real‑time qPCR and at the protein level, by immunohistochemistry, and to examine its correlation, if any, with patient outcome. Five Grade I, 14 Grade II, 18 Grade III, 72 Grade IV samples and 13 normal brain controls were included. We did not find any clear pattern of regulation at the RNA level with tumor grade. The RNA expression levels however, correlated to a large extent with the nuclear $(AP‑2\alpha)$ staining in these samples (72.09%; 31/43). Further, we did not find a complete loss of nuclear $(AP‑2\alpha)$ expression in the higher grades, in contrast to previous reports. Interestingly, we found cytoplasmic $(AP‑2\alpha)$ expression in a majority of higher grade astrocytomas (Grade IV—85%; 33/39 and Grade III—74%; 14/19) in comparison to lower grades (Grade I—0%; 0/5 and Grade II—37.5%; 3/8) suggesting that the translocation of this protein from the nucleus to the cytoplasm may be responsible for the increased malignancy. The nuclear expression in these grades was found to be concomitantly reduced. Within GBMs, we found that decreased nuclear expression was indicative of a better prognosis. The striking observation was the shift in localization of this protein from the nucleus to the cytoplasm with increasing tumor grade, pointing to a crucial role for this transcription factor in the progression of astrocytomas.

• Apoptosis induction by activator Protein 2\alpha involves transcriptional repression of Bcl-2

  Authors: Narendra Wajapeyee, Ramona Britto, Halasahalli M Ravishankar, Kumaravel Somasundar

  Activator protein 2 alpha ( AP-2 alpha) induces cytotoxicity by inducing cell cycle arrest and apoptosis. In this study we investigated the mechanism of apoptosis induction by AP-2 alpha. We foundActivator protein 2 alpha ( AP-2 alpha) induces cytotoxicity by inducing cell cycle arrest and apoptosis. In this study we investigated the mechanism of apoptosis induction by AP-2 alpha. We found that AP-2 alpha induced apoptosis efficiently in cells treated with benzyloxycarbonyl-IETD-fluoromethyl ketone or FADD silenced cells but failed to do so in benzyloxycarbonyl- EHD-fluoromethyl ketone-treated or apoptosis protease activation factor-1 ( Apaf1)-silenced cells, suggesting the central role of mitochondria in AP-2 alpha-induced apoptosis. In good correlation, cells overexpressing AP-2 alpha showed a reduction in mitochondrial membrane potential ( Delta Psi(m)), cytochrome c and Smac/DIABLOm release into cytosol, and Bax translocation into mitochondria. We found that the pro-apoptotic protein Bax is important for AP-2 alpha-induced apoptosis as adenovirus AP2 failed to induce apoptosis in HCT116 Bax(-/-) cells. However, we found the IAP ( inhibitor of apoptosis) inhibitor Smac/DIABLO may have a limited role in AP-2 alpha-induced apoptosis as we found the IAP member Survivin down-regulated by AP-2 alpha. Although the total Bax level remains unaltered, we found a time-dependent increase in the activated form of Bax in adenovirus AP2-infected cells. In addition, we show that AP-2 alpha transcriptionally represses Bcl-2 by binding to its promoter both in vitro and in vivo and that this is essential for AP-2 alpha- nduced apoptosis as ectopic expression of Bcl-2 efficiently inhibited apoptosis induced by AP-2 alpha. Furthermore, we show that chemotherapy-induced endogenous AP-2 alpha down-regulates Bcl-2 and induces apoptosis in an AP-2 alpha-dependent manner. Moreover, we demonstrate that inhibition of okadaic acid or staurosporine-sensitive pathways in AP-2 alpha overexpressing breast cancer cells resulted in AP-2 alpha-dependent apoptosis induction. These results suggest that AP-2 alpha induces apoptosis by down-regulating Bcl-2 and utilizing a bax/cytochrome c/Apaf1/caspase 9-dependent mitochondrial pathway.