Recent genome-wide mapping of the mammalian replication origins has suggested the role of transcr... more Recent genome-wide mapping of the mammalian replication origins has suggested the role of transcriptional regulatory elements in origin activation. However, the nature of chromatin modifications associated with such trans-factors or epigenetic marks imprinted on cis-elements during the spatio-temporal regulation of replication initiation remains enigmatic. To unveil the molecular underpinnings, we studied the human lamin B2 origin that spatially overlaps with TIMM 13 promoter. We observed an early G 1 -specific occupancy of c-Myc that facilitated the loading of mini chromosome maintenance protein (MCM) complex during subsequent mid-G 1 phase rather stimulating TIMM 13 gene expression. Investigations on the Myc-induced downstream events suggested a direct interaction between c-Myc and histone methyltransferase mixed-lineage leukemia 1thatimpartedhistone H3K4me3 mark essential for both recruitment of acetylase complex HBO1 and hyperacetylation of histone H4. Contemporaneously, the nucleosome remodeling promoted the loading of MCM proteins at the origin. These chromatin modifications were under the tight control of active demethylation of E-box as evident from methylation profiling. The active demethylation was mediated by the Teneleven translocation (TET)-thymine DNA glycosylasebase excision repair (BER) pathway, which facilitated spatio-temporal occupancy of Myc. Intriguingly, the genome-wide 43% occurrence of E-box among the human origins could support our hypothesis that epigenetic control of E-box could be a molecular switch for the licensing of early replicating origins.
Background : The introduction of transgenic technology has made it possible to study the steps of... more Background : The introduction of transgenic technology has made it possible to study the steps of carcinogenesis and directly establish the link between viral subgenomic fragments and specific types of cancer. Research directed at hepatitis B virus (HBV)-related carcinogenesis has benefited from this technology. We present a detailed pathological evaluation of the sequential steps of hepatocarcinogenesis in a hepatitis B 'x' (HBx) transgenic mouse model. In this model, the transgene incorporates the region encoding amino acids 58-154 of the HBV X protein and the murine c-myc gene. This model demonstrated changes in the liver from birth with foci of multicentric dysplasia evolving into nodules and overt hepatocellular carcinoma between 20 and 28 weeks. Methods and Results : The hepatocytes were mitotically active and showed increased proliferative capacity soon after birth, with exponential increase thereafter. This was accompanied by a high rate of apoptosis, which later declined as the tumors developed. Other functional and immunophenotypic characteristics included a high c-myc expression in the neoplastic lesions, no alteration in p53 expression, and no alteration in the expression of hepatic enzymes except for diffuse expression of succinic dehydrogenase. Conclusion : The entire process illustrates the disturbances of cell growth and death because of the collaborative influence of HBx and c-myc genes that result in the development of hepatocellular carcinoma after a prolonged latent period.
E3 ubiquitin ligases have been implicated in the ubiquitination and proteasome-mediated degradati... more E3 ubiquitin ligases have been implicated in the ubiquitination and proteasome-mediated degradation of several key regulators of cell cycle. Owing to their pleotropic behavior, E3 ubiquitin ligases are tightly regulated both at transcriptional and post-translational levels. The E3 ubiquitin ligase TRUSS (tumor necrosis factor receptor-associated ubiquitous scaffolding and signaling protein) which negatively regulates c-Myc, are found down-regulated in most human cancer cell lines. However, the mechanism of regulation of intracellular levels of TRUSS remains elusive. Here we show that TRUSS is expressed majorly during the G1 phase of cell cycle and its level starts to decline with the expression of S-phase specific E3 ligase Skp2. Enforced expression of Skp2 led to a marked increase in the ubiquitination of TRUSS after its phosphorylation by GSK3β and followed by rapid proteolytic degradation. Our coimmunoprecipitation studies suggested a direct interaction between Skp2 and TRUSS through the LRR motif of Skp2. Interestingly, the human tumor samples that exhibited elevated expression of Skp2, showed relatively poor expression of TRUSS. Further, enforced expression of HBx, the oncoprotein of Hepatitis B virus which is known to stabilize c-Myc and enhance its oncogenic potential, led to the intracellular accumulation of TRUSS as well as c-Myc. Apparently, HBx also interacted with TRUSS which negatively impacted the TRUSS-c-Myc and TRUSS-Skp2 interactions leading to stabilization of TRUSS. Thus, the present study suggests that TRUSS is a novel substrate of E3 ligase Skp2 and that disruption of TRUSS-Skp2 interaction by viral oncoproteins could lead to pathophysiological sequelae.
Background: In Traditional Tibetan medicine, Yukyung Karne has been used for the treatment of ova... more Background: In Traditional Tibetan medicine, Yukyung Karne has been used for the treatment of ovarian cancer. Though Yukyung Karne has been reported to be clinically effective, the molecular mechanism of its anti-metstatic action remains elusive. Methods: The cytotoxic property of Yukyung Karne was evaluated by crystal violet staining while its ability to induce ceramide production was analyzed by sphingomyelinase assay. The anti-metastatic property was investigated using adhesion, invasion, migration and colony formation assays. The effect of Yukyung Karne on the expression of extracellular matrix components, and epithelial and mesenchymal markers were evaluated by confocal microscopy and western blotting. Results: Yukyung Karne exhibited a strong anti-metastatic property by significantly reducing the invasion, migration and colony formation ability of ovarian cancer cells. Besides it inhibited the levels of biomarkers involved in epithelial to mesenchymal transition such as down-regulation of vimentin and N-cadherin and up-regulation of epithelial E-cadherin. Yukyung Karne also induced the neutral sphingomyelinase II (nSMNaseII) enzyme activity that is known to hydrolyze sphingomyelins into pro-apoptotic intracellular molecule ceramide. Conclusions: The study provides some compelling evidences supporting the anti-metastatic potential of Yukyung Karne which strongly suggests its possible usage as a promising alternative medicine. Thus, Yukyung Karne may be used as an anticance
Nucleolar assembly begins at the early G1 phase of the cell cycle and is a hub of ribosomal DNA t... more Nucleolar assembly begins at the early G1 phase of the cell cycle and is a hub of ribosomal DNA transcription and rRNA biosynthesis. The newlyformed rRNAs together with ribosomal proteins (RPs) constitute the building block of the ribosomal machinery. Although RPs play a major role in protein biosynthesis, their own regulation and expression is rather poorly understood. In the present study, we investigated the regulation of RP genes RPS27a, RPS24, RPS6, RPL9 and RPL4 in synchronized mammalian cell culture. Quantitative RT-PCR analysis indicated their expression during the mid to late G1 phase, whereas the rRNA genes were expressed during the early G1 phase of the cell cycle. The promoter reporter analysis of the RPS27a gene revealed that it could be synergistically stimulated by the transcription factors specificity protein 1 (Sp1) and cAMP response element-binding protein (CREB). However, E2F transcription factor 1 (E2F1) appeared to negatively regulate gene expression. Chromatin immunoprecipitation studies confirmed the promoter occupancy of Sp1, CREB and E2F1. Although Sp1 and CREB binding enhanced the promoter occupancy of histone acetyltransferases PCAF, p300 and CREB binding protein, E2F1 facilitated the recruitment of histone deacetylases. Both acetylation (histone H4 pan-acetyl, histone H3 acetyl Lys 14) and methylation (histone H3 trimethyl Lys 9) marks were observed in the RPS27a promoter region, suggesting their important regulatory role in gene expression. Because the promoter regions of most RP genes are well conserved, we propose that their orchestrated regulation and synthesis during the cell cycle facilitates ribosome biogenesis.
Many proteins rely on disulfide bonds formed between pairs of cysteines for the stability of thei... more Many proteins rely on disulfide bonds formed between pairs of cysteines for the stability of their folded state and to keep regulatory control over their functions. The hepatitis B virus-encoded HBx oncoprotein is known to perform an overwhelming array of functions in the cell and has been implicated in the development of hepatocellular carcinoma. However, its structure has not been elucidated. HBx carries nine conserved cysteine residues that have proven to be crucial for its various functions. However, the status of disulfide bonds between the cysteine residues reported in previous studies remains discrepant because of the use of refolded recombinant HBx that may contain non-native disulfides. Now we have determined the disulfide linkages in soluble and biologically active recombinant maltose binding protein-HBx fusion protein using matrix-assisted laser desorption ionization time-of-flight mass spectrometry. We report four disulfide linkages in HBx protein, viz., between Cys(7) a...
Ethnopharmacological relevance: Butea monosperma (Lam.) (Fabaceae) popularly known as 'flame of t... more Ethnopharmacological relevance: Butea monosperma (Lam.) (Fabaceae) popularly known as 'flame of the forest' has been widely used in the traditional Indian medical system of 'Ayurveda' for the treatment of a variety of ailments including liver disorders. Aim of the study: To evaluate the antioxidative, anti-inflammatory, hepatoprotective and anti-cancer activities of the aqueous extract of Butea monosperma flowers. Materials and methods: Dried flowers of Butea monosperma were extracted with water. The extract was tested for its anti-proliferative, pro-apoptotic and anti-carcinogenic effects in hepatoma cell lines. The chemopreventive and anti-angiogenic effects of the extract were evaluated by its daily oral administration in a HBV-related X15-myc mouse model of hepatocellular carcinoma (HCC). Results: Treatment with the aqueous extract inhibited cell proliferation and accumulation of cells in G1 phase. This was accompanied by a marked reduction in the levels of activated Erk1/2 and SAPK/JNK and induction of apoptotic cell death. Oral administration of the extract in transgenic mice conferred hepatoprotection as is evident from normal serum ALT levels and improved liver histopathology and lowered serum VEGF level. Conclusions: The ability of aqueous extract of Butea monosperma flowers to impose growth arrest and trigger pro-apoptotic death in cell culture strongly correlated with its strong chemopreventive effect in vivo when given orally.
The pleiotropic HBx oncoprotein of hepatitis B virus is well known to promote the expression of r... more The pleiotropic HBx oncoprotein of hepatitis B virus is well known to promote the expression of ribosomal RNAs and several host proteins that are known to support the development and progression of hepatocellular carcinoma (HCC). While overexpression of the nucleolar phosphoprotein, nucleophosmin (NPM), correlates with HCC progression, its upregulation by viral HBx and the resulting impact on perturbed nucleolar functions remain enigmatic. The present study shows that HBx up-regulates NPM levels and hijacks its functions to promote cellular proliferation. We found that HBx expression stabilizes NPM through post-translational modifications. Enhanced CDK2-mediated phosphorylation of NPM at Thr199 upon HBx expression prevented its proteolytic cleavage and provided resistance to apoptosis. Further, HBx directly interacted with the C-terminal domain of NPM and got translocated into the nucleolus where it facilitated the recruitment of RNA polymerase I transcriptional machinery onto the rDNA promoter. Our results indicate that HBx enhances rDNA transcription via a novel regulatory mechanism involving acetylation of NPM and the subsequent depletion of histones from the rDNA promoter. Enhanced production of ribosomal RNA resulting from co-expression of HBx and NPM promoted ribosome biogenesis, cellular proliferation and transformation. Taken together, our study strongly suggests an important role of NPM in mediating the oncogenic effects of HBx and the corresponding nucleolar perturbations induced by this viral oncoprotein.
Metazoans utilize multiple origins of replication in order to replicate their vast genome faithfu... more Metazoans utilize multiple origins of replication in order to replicate their vast genome faithfully and expeditiously during each cell cycle. However, lesser known are the salient features defining these origins and their mechanism of selection for replication process. Here, we provide burgeoning evidences which suggest that transcription factors indeed play a major role in facilitating licensing of origins for firing during the S phase.
Background: The HBx oncoprotein of hepatitis B virus has been implicated in the development and p... more Background: The HBx oncoprotein of hepatitis B virus has been implicated in the development and progression of hepatocellular carcinoma (HCC). HBx engages multiple signalling and growth-promoting pathways to induce cell proliferation and enhance ribosome biogenesis. Interestingly, the levels of Upstream Binding Factor (UBF) required for rDNA transcription and ribosome biogenesis are found elevated in the HCC patients. However, the molecular mechanism of UBF overexpression under the HBx microenvironment and consequent cell transformation remains elusive. Methods: The UBF gene expression was investigated after co-expressing HBx in immortalized human hepatocytes (IHH) and human hepatoma Huh7 cells. Gene expression analysis involved estimation of mRNA level by real-time PCR, western blotting of protein, chromatin immune-precipitation assay, BrdU incorporation assay and soft agar colony formation assay. UBF expression was also investigated in an HBx transgenic mouse model of HCC to get a better mechanistic insight under more physiological conditions. Results: Ectopic expression of HBx in IHH as well as Huh7 cells led to a marked increase in UBF expression both at mRNA and protein levels. Elevated levels of UBF were also observed in the hepatic tumors of HBx transgenic mice. Our ChIP studies revealed a marked increase in the occupancy of c-Myc on the UBF gene promoter in the presence of HBx and increase in its transcription. Enhanced UBF expression under the HBx microenvironment led to a marked increase in cell proliferation and transformation of IHH cells. Conclusions: Our study provides some compelling evidences in support of HBx-mediated increase in UBF levels that abets oncogenic onslaught in hepatic cells by increasing rDNA transcription and ribosome biogenesis.
The small ribosomal protein RPS27a is known to play a role in the activation of cellular checkpoi... more The small ribosomal protein RPS27a is known to play a role in the activation of cellular checkpoints via p53 which links ribosome biogenesis to cell cycle progression. Here, we show that RPS27a gene is a direct transcriptional target of p53 and is overexpressed in response to DNA damage. Elevated RPS27a level was associated with increased expression of p53 and its target p21Waf1 gene. The RPS27a activity was specifically inhibited in the presence of a dominant negative mutant of p53. Down-regulation of ectopically expressed RPS27a by RNA interference blocked the activation of p21waf1 in response to DNA damage. Thus, RPS27a appears to be a novel stress sensor in the cell which amplifies p53 response to arrest cell cycle.
Hepatotropic viruses such as Hepatitis B (HBV) and Hepatitis C virus (HCV) are the major etiologi... more Hepatotropic viruses such as Hepatitis B (HBV) and Hepatitis C virus (HCV) are the major etiological agents associated with development of hepatocellular carcinoma (HCC). Progression of HCC is a multistep process that requires sequential or parallel deregulation of oncogenic and tumor suppressive pathways leading to chromosomal instability and neoplastic phenotype. In the recent years, microRNAs (miRNAs) have carved their own niche alongside oncogenes and tumor suppressors, owing to their innate ability to receive and relay multiple signals. Not surprisingly, miRNAs are fast emerging as central player in myriads of malignancies including HCC. MiRNAs are reported to participate in initiation and progression of HCC, as well as have been clinically correlated with risk assessment, disease grade, aggressiveness and prognosis. Despite extensive data available on the role of miRNAs in HCC, there is a pressing need to integrate and evaluate these datasets to find its correlation, if any, with causal agents in order to devise novel interventional modalities. Through this review, we attempt to bridge the gap by consolidating the current knowledge and concepts in the field of HCC-related miRNAs with special emphasis on HBV and HCV. Further, we assess the potential of common as well as unique signatures that may be useful in developing novel biomarkers and therapeutics.
Notoriously recognized as the principal cause of mortality among women worldwide, breast cancer c... more Notoriously recognized as the principal cause of mortality among women worldwide, breast cancer calls for an immediate redressal in order to devise combative strategies. Currently, Northern America and Western Europe experience more than half of the global burden of breast cancer. However, as recently reported by International Agency for Research on Cancer (IARC), rapid societal and economic transitions herald an epidemiological shift in the incidence, with more low income countries projected for an increased burden of breast cancer. Although the incidence rates remain higher in more developed countries, mortality and morbidity are higher in less developed regions, attributable to late detection and lack of access to advanced medical amenities. Regardless of epidemiological prevalence, the etiological determinants of breast cancer development remain common and include age, family history, genetic risk factors such as BRCA1 and BRCA2 mutations and hormonal risk factors. Although viruses have been etiologically associated with many cancers, a viral etiology to breast cancer is at best speculative. There is conflicting evidence of roles of viruses such as human mammary tumor virus, human papillomavirus, Epstein-Barr virus, human cytomegalovirus, herpes simplex virus and measles virus in breast cancer development. Nonetheless, a recent development that levels of mutagenic antiviral enzyme APOBEC3B are elevated in a majority of breast cancers warrants a re-visit to the role of viruses in breast cancer pathogenesis. Keeping in view the current scenario, there is an urgency to assess the worldwide trends and risk factors for prediction of future scenarios with the ultimate goal of developing effective, affordable and prioritized approaches for breast cancer control.
The HBx oncoprotein of hepatitis B Virus has been accredited as one of the protagonist in driving... more The HBx oncoprotein of hepatitis B Virus has been accredited as one of the protagonist in driving hepatocarcinogenesis. HBx exerts its influence over the cell cycle progression by potentiating the activity of cyclin A/E-CDK2 complex, the Cyclin A partner of which is a well-known target of cellular deubiquitinase USP37. In the present study, we observed the intracellular accumulation of cyclin A and USP37 proteins under the HBx microenvironment. Flow cytometry analysis of the HBx-expressing cells showed deregulation of cell cycle apparently due to the enhanced gene expression and stabilization of USP37 protein and deubiquitination of Cyclin A by USP37. Our co-immunoprecipitation and confocal microscopic studies suggested a direct interaction between USP37 and HBx. This interaction promoted the translocation of USP37 outside the nucleus and prevented its association and ubiquitination by E3 ubiquitin ligases - APC/CDH1 and SCF/β-TrCP. Thus, HBx seems to control the cell cycle progression via the cyclin A-CDK2 complex by regulating the intracellular distribution and stability of deubiquitinase USP37.
Background: Yukyung karne (YK) is a traditional Tibetan formulation used for many centuries for t... more Background: Yukyung karne (YK) is a traditional Tibetan formulation used for many centuries for the treatment of ovarian cancer. However, the pharmacological basis of its anticancer property is not well understood. In the present study, the anticancer property of YK was investigated in cell culture. Methods: The growth inhibition property of YK was evaluated in SKOV6, IHH, HepG2 and HEK293 cell lines using MTT assay. The pro-apoptotic activity of drug was analyzed by terminal deoxynuleotidyl transferase dUTP nick end labeling (TUNEL) and DNA fragmentation assays. Confocal microscopy was used to show the release of cytochrome c and its co-localization with mitochondria with the help of dsRed mitotracker in SKOV6 cells. The inhibition in cell proliferation was also visualized by confocal microscopy after BrDU incorporation. The activation of tumor suppressor p53 was evaluated by Western blotting while the VEGF level in culture supernatant was measured by a colorimetric method. Results: YK specifically and efficiently induced apoptotic killing of the human ovarian cancer SKOV6 cells as indicated by increased DNA fragmentation and nick end DNA labeling. Confocal microscopy suggested inhibition of cell proliferation and increase in cytochrome c release via perturbation in mitochondrial membrane potential (m). Further, YK up-regulated the expression of tumor suppressor p53 and key cyclin-dependent kinase inhibitor p21, and inhibited VEGF secretion by cells. Interestingly, YK also exhibited a synergy with paclitaxel which a well-known anti-cancer therapeutic drug. Conclusions: The pharmacological properties of YK to impose growth arrest and trigger pro-apoptotic death in cells amply justifies its usage in primary as well as adjunct therapy for ovarian cancer.
Viruses are well known for their ability to hijack and manipulate the host cellular machinery to ... more Viruses are well known for their ability to hijack and manipulate the host cellular machinery to ensure immune evasion, viral survival and pathogenesis. Most animal viruses exhibit exclusive tropism and thus, infect only specific target cells. However, reports on the existence of virions and viral components in non-target cells suggest alternative mechanisms of viral spread. Studies on microvesicles and exosomes promise to provide justification for the presence of viruses at unrelated cell types. Exosomes have attracted the attention of not only cell biologist but also virologist as these vesicles can transport and deliver bioactive information (RNA, proteins, microRNA etc. including virus specific components from infected cells) to unrelated cell types and have the potential to regulate target cell function. Recent studies suggest that viruses can manipulate and hijack the exosome biogenesis and secretory pathway to manipulate the host microenvironment, evade immune response and increase viral accessibility. Here, we review the existing literature on viral interference and exploitation of exosome secretory mechanisms and correlate it with the increased virulence and spread of viruses in the host. Further, we discuss the prospects of exosomes as emerging biomarkers for virus induced pathology, potential of exosomes as delivery vehicles and also the new perspective to viral mediated pathogenesis.
The role of viral infection in cancer was established
towards the beginning of 20th century. The ... more The role of viral infection in cancer was established towards the beginning of 20th century. The study of tumour viruses, their oncogenes and different mechanisms employed by these viruses to subvert the growth-suppressive and pro-apoptotic functions of host tumour suppressor genes has laid the foundation of cancer biology. The human tumour viruses induce malignancies after a prolonged latency and in conjunction with other environmental and host factors. The eight known human tumour viruses contribute to nearly 10–15% of the cancers worldwide. Advancements in research on virus-related cancers offer a plethora of opportunities to fight cancer by preventing viral spread through vaccination and use of antivirals. Besides, recent developments on viral oncogenic mechanisms should allow development of novel and targeted approaches for control and treatment of virus-associated human cancers.
In this technologically savvy world, we witness mushrooming of sophisticated lines of softwares t... more In this technologically savvy world, we witness mushrooming of sophisticated lines of softwares that are extensively used for predicting epitopes towards developing vaccines and therapeutics. However, most softwares also generate bulky junk data along with data of interest. Here we devised a combined usage of B cell epitope prediction softwares - ABCpred along with additional online tools such as CLUSTAL W and Sequence Manipulation Suite for stringent selection of B cell epitopes. Dengue was used as model disease since the present diagnostic kits give false positives owing to cross-reactivity with other flaviviruses. The in silico predicted 240 unique B cell epitopes of dengue viruses were reduced to 87 serotype-specific epitopes using combination of softwares. Three best ranking epitopes of each dengue serotype were functionally validated by cloning corresponding DNA sequences in the pCBP vector and expressing these epitopes as calcium-binding protein fusion proteins. Western blot analysis and indirect ELISAs of purified fusion proteins confirmed that nearly fifty per cent of the epitopes were in close agreement with the predictions. Thus, stringent selection of epitopes using a combination of prediction softwares is likely to identify a compact library of B cell epitopes useful for developing more specific diagnostics in future.
Nucleolar assembly begins at the early G1 phase of cell cycle and is a hub of ribosomal DNA trans... more Nucleolar assembly begins at the early G1 phase of cell cycle and is a hub of ribosomal DNA transcription and ribosomal RNA (rRNA) biosynthesis. The newly formed rRNAs together with ribosomal proteins constitute the building block of the ribosomal machinery. Although ribosomal proteins play a major role in protein biosynthesis, their own regulation and expression is rather poorly understood. Here, we investigated the regulation ribosomal protein genes RPS27a, RPS24, RPS6, RPL9 and RPL4 in synchronized mammalian cell culture. The real-time quantitative PCR analysis indicated their expression during the mid to late G1 phase whereas the ribosomal RNA genes were expressed during the early G1 phase of cell cycle. The promoter reporter analysis of the RPS27a gene revealed that it could be synergistically stimulated by the transcription factors Sp1 and CREB. However, E2F1 appeared to negatively regulate the gene expression. The chromatin immunoprecipitation studies confirmed the promoter occupancy of Sp1, CREB and E2F1. While Sp1 and CREB binding enhanced the promoter occupancy of histone acetyltransferases PCAF, p300 and CBP, E2F1 facilitated the recruitment of histone deacetylases. Both acetylation (H4ac, H3K14ac) and methylation (H3K9me3) marks were observed in the RPS27a promoter region suggesting their important regulatory role in gene expression. Since the promoter regions of most ribosomal protein genes are well conserved, we propose their orchestrated regulation and synthesis during the cell cycle facilitates ribosome biogenesis.
Recent genome-wide mapping of the mammalian replication origins has suggested the role of transcr... more Recent genome-wide mapping of the mammalian replication origins has suggested the role of transcriptional regulatory elements in origin activation. However, the nature of chromatin modifications associated with such trans-factors or epigenetic marks imprinted on cis-elements during the spatio-temporal regulation of replication initiation remains enigmatic. To unveil the molecular underpinnings, we studied the human lamin B2 origin that spatially overlaps with TIMM 13 promoter. We observed an early G 1 -specific occupancy of c-Myc that facilitated the loading of mini chromosome maintenance protein (MCM) complex during subsequent mid-G 1 phase rather stimulating TIMM 13 gene expression. Investigations on the Myc-induced downstream events suggested a direct interaction between c-Myc and histone methyltransferase mixed-lineage leukemia 1thatimpartedhistone H3K4me3 mark essential for both recruitment of acetylase complex HBO1 and hyperacetylation of histone H4. Contemporaneously, the nucleosome remodeling promoted the loading of MCM proteins at the origin. These chromatin modifications were under the tight control of active demethylation of E-box as evident from methylation profiling. The active demethylation was mediated by the Teneleven translocation (TET)-thymine DNA glycosylasebase excision repair (BER) pathway, which facilitated spatio-temporal occupancy of Myc. Intriguingly, the genome-wide 43% occurrence of E-box among the human origins could support our hypothesis that epigenetic control of E-box could be a molecular switch for the licensing of early replicating origins.
Background : The introduction of transgenic technology has made it possible to study the steps of... more Background : The introduction of transgenic technology has made it possible to study the steps of carcinogenesis and directly establish the link between viral subgenomic fragments and specific types of cancer. Research directed at hepatitis B virus (HBV)-related carcinogenesis has benefited from this technology. We present a detailed pathological evaluation of the sequential steps of hepatocarcinogenesis in a hepatitis B 'x' (HBx) transgenic mouse model. In this model, the transgene incorporates the region encoding amino acids 58-154 of the HBV X protein and the murine c-myc gene. This model demonstrated changes in the liver from birth with foci of multicentric dysplasia evolving into nodules and overt hepatocellular carcinoma between 20 and 28 weeks. Methods and Results : The hepatocytes were mitotically active and showed increased proliferative capacity soon after birth, with exponential increase thereafter. This was accompanied by a high rate of apoptosis, which later declined as the tumors developed. Other functional and immunophenotypic characteristics included a high c-myc expression in the neoplastic lesions, no alteration in p53 expression, and no alteration in the expression of hepatic enzymes except for diffuse expression of succinic dehydrogenase. Conclusion : The entire process illustrates the disturbances of cell growth and death because of the collaborative influence of HBx and c-myc genes that result in the development of hepatocellular carcinoma after a prolonged latent period.
E3 ubiquitin ligases have been implicated in the ubiquitination and proteasome-mediated degradati... more E3 ubiquitin ligases have been implicated in the ubiquitination and proteasome-mediated degradation of several key regulators of cell cycle. Owing to their pleotropic behavior, E3 ubiquitin ligases are tightly regulated both at transcriptional and post-translational levels. The E3 ubiquitin ligase TRUSS (tumor necrosis factor receptor-associated ubiquitous scaffolding and signaling protein) which negatively regulates c-Myc, are found down-regulated in most human cancer cell lines. However, the mechanism of regulation of intracellular levels of TRUSS remains elusive. Here we show that TRUSS is expressed majorly during the G1 phase of cell cycle and its level starts to decline with the expression of S-phase specific E3 ligase Skp2. Enforced expression of Skp2 led to a marked increase in the ubiquitination of TRUSS after its phosphorylation by GSK3β and followed by rapid proteolytic degradation. Our coimmunoprecipitation studies suggested a direct interaction between Skp2 and TRUSS through the LRR motif of Skp2. Interestingly, the human tumor samples that exhibited elevated expression of Skp2, showed relatively poor expression of TRUSS. Further, enforced expression of HBx, the oncoprotein of Hepatitis B virus which is known to stabilize c-Myc and enhance its oncogenic potential, led to the intracellular accumulation of TRUSS as well as c-Myc. Apparently, HBx also interacted with TRUSS which negatively impacted the TRUSS-c-Myc and TRUSS-Skp2 interactions leading to stabilization of TRUSS. Thus, the present study suggests that TRUSS is a novel substrate of E3 ligase Skp2 and that disruption of TRUSS-Skp2 interaction by viral oncoproteins could lead to pathophysiological sequelae.
Background: In Traditional Tibetan medicine, Yukyung Karne has been used for the treatment of ova... more Background: In Traditional Tibetan medicine, Yukyung Karne has been used for the treatment of ovarian cancer. Though Yukyung Karne has been reported to be clinically effective, the molecular mechanism of its anti-metstatic action remains elusive. Methods: The cytotoxic property of Yukyung Karne was evaluated by crystal violet staining while its ability to induce ceramide production was analyzed by sphingomyelinase assay. The anti-metastatic property was investigated using adhesion, invasion, migration and colony formation assays. The effect of Yukyung Karne on the expression of extracellular matrix components, and epithelial and mesenchymal markers were evaluated by confocal microscopy and western blotting. Results: Yukyung Karne exhibited a strong anti-metastatic property by significantly reducing the invasion, migration and colony formation ability of ovarian cancer cells. Besides it inhibited the levels of biomarkers involved in epithelial to mesenchymal transition such as down-regulation of vimentin and N-cadherin and up-regulation of epithelial E-cadherin. Yukyung Karne also induced the neutral sphingomyelinase II (nSMNaseII) enzyme activity that is known to hydrolyze sphingomyelins into pro-apoptotic intracellular molecule ceramide. Conclusions: The study provides some compelling evidences supporting the anti-metastatic potential of Yukyung Karne which strongly suggests its possible usage as a promising alternative medicine. Thus, Yukyung Karne may be used as an anticance
Nucleolar assembly begins at the early G1 phase of the cell cycle and is a hub of ribosomal DNA t... more Nucleolar assembly begins at the early G1 phase of the cell cycle and is a hub of ribosomal DNA transcription and rRNA biosynthesis. The newlyformed rRNAs together with ribosomal proteins (RPs) constitute the building block of the ribosomal machinery. Although RPs play a major role in protein biosynthesis, their own regulation and expression is rather poorly understood. In the present study, we investigated the regulation of RP genes RPS27a, RPS24, RPS6, RPL9 and RPL4 in synchronized mammalian cell culture. Quantitative RT-PCR analysis indicated their expression during the mid to late G1 phase, whereas the rRNA genes were expressed during the early G1 phase of the cell cycle. The promoter reporter analysis of the RPS27a gene revealed that it could be synergistically stimulated by the transcription factors specificity protein 1 (Sp1) and cAMP response element-binding protein (CREB). However, E2F transcription factor 1 (E2F1) appeared to negatively regulate gene expression. Chromatin immunoprecipitation studies confirmed the promoter occupancy of Sp1, CREB and E2F1. Although Sp1 and CREB binding enhanced the promoter occupancy of histone acetyltransferases PCAF, p300 and CREB binding protein, E2F1 facilitated the recruitment of histone deacetylases. Both acetylation (histone H4 pan-acetyl, histone H3 acetyl Lys 14) and methylation (histone H3 trimethyl Lys 9) marks were observed in the RPS27a promoter region, suggesting their important regulatory role in gene expression. Because the promoter regions of most RP genes are well conserved, we propose that their orchestrated regulation and synthesis during the cell cycle facilitates ribosome biogenesis.
Many proteins rely on disulfide bonds formed between pairs of cysteines for the stability of thei... more Many proteins rely on disulfide bonds formed between pairs of cysteines for the stability of their folded state and to keep regulatory control over their functions. The hepatitis B virus-encoded HBx oncoprotein is known to perform an overwhelming array of functions in the cell and has been implicated in the development of hepatocellular carcinoma. However, its structure has not been elucidated. HBx carries nine conserved cysteine residues that have proven to be crucial for its various functions. However, the status of disulfide bonds between the cysteine residues reported in previous studies remains discrepant because of the use of refolded recombinant HBx that may contain non-native disulfides. Now we have determined the disulfide linkages in soluble and biologically active recombinant maltose binding protein-HBx fusion protein using matrix-assisted laser desorption ionization time-of-flight mass spectrometry. We report four disulfide linkages in HBx protein, viz., between Cys(7) a...
Ethnopharmacological relevance: Butea monosperma (Lam.) (Fabaceae) popularly known as 'flame of t... more Ethnopharmacological relevance: Butea monosperma (Lam.) (Fabaceae) popularly known as 'flame of the forest' has been widely used in the traditional Indian medical system of 'Ayurveda' for the treatment of a variety of ailments including liver disorders. Aim of the study: To evaluate the antioxidative, anti-inflammatory, hepatoprotective and anti-cancer activities of the aqueous extract of Butea monosperma flowers. Materials and methods: Dried flowers of Butea monosperma were extracted with water. The extract was tested for its anti-proliferative, pro-apoptotic and anti-carcinogenic effects in hepatoma cell lines. The chemopreventive and anti-angiogenic effects of the extract were evaluated by its daily oral administration in a HBV-related X15-myc mouse model of hepatocellular carcinoma (HCC). Results: Treatment with the aqueous extract inhibited cell proliferation and accumulation of cells in G1 phase. This was accompanied by a marked reduction in the levels of activated Erk1/2 and SAPK/JNK and induction of apoptotic cell death. Oral administration of the extract in transgenic mice conferred hepatoprotection as is evident from normal serum ALT levels and improved liver histopathology and lowered serum VEGF level. Conclusions: The ability of aqueous extract of Butea monosperma flowers to impose growth arrest and trigger pro-apoptotic death in cell culture strongly correlated with its strong chemopreventive effect in vivo when given orally.
The pleiotropic HBx oncoprotein of hepatitis B virus is well known to promote the expression of r... more The pleiotropic HBx oncoprotein of hepatitis B virus is well known to promote the expression of ribosomal RNAs and several host proteins that are known to support the development and progression of hepatocellular carcinoma (HCC). While overexpression of the nucleolar phosphoprotein, nucleophosmin (NPM), correlates with HCC progression, its upregulation by viral HBx and the resulting impact on perturbed nucleolar functions remain enigmatic. The present study shows that HBx up-regulates NPM levels and hijacks its functions to promote cellular proliferation. We found that HBx expression stabilizes NPM through post-translational modifications. Enhanced CDK2-mediated phosphorylation of NPM at Thr199 upon HBx expression prevented its proteolytic cleavage and provided resistance to apoptosis. Further, HBx directly interacted with the C-terminal domain of NPM and got translocated into the nucleolus where it facilitated the recruitment of RNA polymerase I transcriptional machinery onto the rDNA promoter. Our results indicate that HBx enhances rDNA transcription via a novel regulatory mechanism involving acetylation of NPM and the subsequent depletion of histones from the rDNA promoter. Enhanced production of ribosomal RNA resulting from co-expression of HBx and NPM promoted ribosome biogenesis, cellular proliferation and transformation. Taken together, our study strongly suggests an important role of NPM in mediating the oncogenic effects of HBx and the corresponding nucleolar perturbations induced by this viral oncoprotein.
Metazoans utilize multiple origins of replication in order to replicate their vast genome faithfu... more Metazoans utilize multiple origins of replication in order to replicate their vast genome faithfully and expeditiously during each cell cycle. However, lesser known are the salient features defining these origins and their mechanism of selection for replication process. Here, we provide burgeoning evidences which suggest that transcription factors indeed play a major role in facilitating licensing of origins for firing during the S phase.
Background: The HBx oncoprotein of hepatitis B virus has been implicated in the development and p... more Background: The HBx oncoprotein of hepatitis B virus has been implicated in the development and progression of hepatocellular carcinoma (HCC). HBx engages multiple signalling and growth-promoting pathways to induce cell proliferation and enhance ribosome biogenesis. Interestingly, the levels of Upstream Binding Factor (UBF) required for rDNA transcription and ribosome biogenesis are found elevated in the HCC patients. However, the molecular mechanism of UBF overexpression under the HBx microenvironment and consequent cell transformation remains elusive. Methods: The UBF gene expression was investigated after co-expressing HBx in immortalized human hepatocytes (IHH) and human hepatoma Huh7 cells. Gene expression analysis involved estimation of mRNA level by real-time PCR, western blotting of protein, chromatin immune-precipitation assay, BrdU incorporation assay and soft agar colony formation assay. UBF expression was also investigated in an HBx transgenic mouse model of HCC to get a better mechanistic insight under more physiological conditions. Results: Ectopic expression of HBx in IHH as well as Huh7 cells led to a marked increase in UBF expression both at mRNA and protein levels. Elevated levels of UBF were also observed in the hepatic tumors of HBx transgenic mice. Our ChIP studies revealed a marked increase in the occupancy of c-Myc on the UBF gene promoter in the presence of HBx and increase in its transcription. Enhanced UBF expression under the HBx microenvironment led to a marked increase in cell proliferation and transformation of IHH cells. Conclusions: Our study provides some compelling evidences in support of HBx-mediated increase in UBF levels that abets oncogenic onslaught in hepatic cells by increasing rDNA transcription and ribosome biogenesis.
The small ribosomal protein RPS27a is known to play a role in the activation of cellular checkpoi... more The small ribosomal protein RPS27a is known to play a role in the activation of cellular checkpoints via p53 which links ribosome biogenesis to cell cycle progression. Here, we show that RPS27a gene is a direct transcriptional target of p53 and is overexpressed in response to DNA damage. Elevated RPS27a level was associated with increased expression of p53 and its target p21Waf1 gene. The RPS27a activity was specifically inhibited in the presence of a dominant negative mutant of p53. Down-regulation of ectopically expressed RPS27a by RNA interference blocked the activation of p21waf1 in response to DNA damage. Thus, RPS27a appears to be a novel stress sensor in the cell which amplifies p53 response to arrest cell cycle.
Hepatotropic viruses such as Hepatitis B (HBV) and Hepatitis C virus (HCV) are the major etiologi... more Hepatotropic viruses such as Hepatitis B (HBV) and Hepatitis C virus (HCV) are the major etiological agents associated with development of hepatocellular carcinoma (HCC). Progression of HCC is a multistep process that requires sequential or parallel deregulation of oncogenic and tumor suppressive pathways leading to chromosomal instability and neoplastic phenotype. In the recent years, microRNAs (miRNAs) have carved their own niche alongside oncogenes and tumor suppressors, owing to their innate ability to receive and relay multiple signals. Not surprisingly, miRNAs are fast emerging as central player in myriads of malignancies including HCC. MiRNAs are reported to participate in initiation and progression of HCC, as well as have been clinically correlated with risk assessment, disease grade, aggressiveness and prognosis. Despite extensive data available on the role of miRNAs in HCC, there is a pressing need to integrate and evaluate these datasets to find its correlation, if any, with causal agents in order to devise novel interventional modalities. Through this review, we attempt to bridge the gap by consolidating the current knowledge and concepts in the field of HCC-related miRNAs with special emphasis on HBV and HCV. Further, we assess the potential of common as well as unique signatures that may be useful in developing novel biomarkers and therapeutics.
Notoriously recognized as the principal cause of mortality among women worldwide, breast cancer c... more Notoriously recognized as the principal cause of mortality among women worldwide, breast cancer calls for an immediate redressal in order to devise combative strategies. Currently, Northern America and Western Europe experience more than half of the global burden of breast cancer. However, as recently reported by International Agency for Research on Cancer (IARC), rapid societal and economic transitions herald an epidemiological shift in the incidence, with more low income countries projected for an increased burden of breast cancer. Although the incidence rates remain higher in more developed countries, mortality and morbidity are higher in less developed regions, attributable to late detection and lack of access to advanced medical amenities. Regardless of epidemiological prevalence, the etiological determinants of breast cancer development remain common and include age, family history, genetic risk factors such as BRCA1 and BRCA2 mutations and hormonal risk factors. Although viruses have been etiologically associated with many cancers, a viral etiology to breast cancer is at best speculative. There is conflicting evidence of roles of viruses such as human mammary tumor virus, human papillomavirus, Epstein-Barr virus, human cytomegalovirus, herpes simplex virus and measles virus in breast cancer development. Nonetheless, a recent development that levels of mutagenic antiviral enzyme APOBEC3B are elevated in a majority of breast cancers warrants a re-visit to the role of viruses in breast cancer pathogenesis. Keeping in view the current scenario, there is an urgency to assess the worldwide trends and risk factors for prediction of future scenarios with the ultimate goal of developing effective, affordable and prioritized approaches for breast cancer control.
The HBx oncoprotein of hepatitis B Virus has been accredited as one of the protagonist in driving... more The HBx oncoprotein of hepatitis B Virus has been accredited as one of the protagonist in driving hepatocarcinogenesis. HBx exerts its influence over the cell cycle progression by potentiating the activity of cyclin A/E-CDK2 complex, the Cyclin A partner of which is a well-known target of cellular deubiquitinase USP37. In the present study, we observed the intracellular accumulation of cyclin A and USP37 proteins under the HBx microenvironment. Flow cytometry analysis of the HBx-expressing cells showed deregulation of cell cycle apparently due to the enhanced gene expression and stabilization of USP37 protein and deubiquitination of Cyclin A by USP37. Our co-immunoprecipitation and confocal microscopic studies suggested a direct interaction between USP37 and HBx. This interaction promoted the translocation of USP37 outside the nucleus and prevented its association and ubiquitination by E3 ubiquitin ligases - APC/CDH1 and SCF/β-TrCP. Thus, HBx seems to control the cell cycle progression via the cyclin A-CDK2 complex by regulating the intracellular distribution and stability of deubiquitinase USP37.
Background: Yukyung karne (YK) is a traditional Tibetan formulation used for many centuries for t... more Background: Yukyung karne (YK) is a traditional Tibetan formulation used for many centuries for the treatment of ovarian cancer. However, the pharmacological basis of its anticancer property is not well understood. In the present study, the anticancer property of YK was investigated in cell culture. Methods: The growth inhibition property of YK was evaluated in SKOV6, IHH, HepG2 and HEK293 cell lines using MTT assay. The pro-apoptotic activity of drug was analyzed by terminal deoxynuleotidyl transferase dUTP nick end labeling (TUNEL) and DNA fragmentation assays. Confocal microscopy was used to show the release of cytochrome c and its co-localization with mitochondria with the help of dsRed mitotracker in SKOV6 cells. The inhibition in cell proliferation was also visualized by confocal microscopy after BrDU incorporation. The activation of tumor suppressor p53 was evaluated by Western blotting while the VEGF level in culture supernatant was measured by a colorimetric method. Results: YK specifically and efficiently induced apoptotic killing of the human ovarian cancer SKOV6 cells as indicated by increased DNA fragmentation and nick end DNA labeling. Confocal microscopy suggested inhibition of cell proliferation and increase in cytochrome c release via perturbation in mitochondrial membrane potential (m). Further, YK up-regulated the expression of tumor suppressor p53 and key cyclin-dependent kinase inhibitor p21, and inhibited VEGF secretion by cells. Interestingly, YK also exhibited a synergy with paclitaxel which a well-known anti-cancer therapeutic drug. Conclusions: The pharmacological properties of YK to impose growth arrest and trigger pro-apoptotic death in cells amply justifies its usage in primary as well as adjunct therapy for ovarian cancer.
Viruses are well known for their ability to hijack and manipulate the host cellular machinery to ... more Viruses are well known for their ability to hijack and manipulate the host cellular machinery to ensure immune evasion, viral survival and pathogenesis. Most animal viruses exhibit exclusive tropism and thus, infect only specific target cells. However, reports on the existence of virions and viral components in non-target cells suggest alternative mechanisms of viral spread. Studies on microvesicles and exosomes promise to provide justification for the presence of viruses at unrelated cell types. Exosomes have attracted the attention of not only cell biologist but also virologist as these vesicles can transport and deliver bioactive information (RNA, proteins, microRNA etc. including virus specific components from infected cells) to unrelated cell types and have the potential to regulate target cell function. Recent studies suggest that viruses can manipulate and hijack the exosome biogenesis and secretory pathway to manipulate the host microenvironment, evade immune response and increase viral accessibility. Here, we review the existing literature on viral interference and exploitation of exosome secretory mechanisms and correlate it with the increased virulence and spread of viruses in the host. Further, we discuss the prospects of exosomes as emerging biomarkers for virus induced pathology, potential of exosomes as delivery vehicles and also the new perspective to viral mediated pathogenesis.
The role of viral infection in cancer was established
towards the beginning of 20th century. The ... more The role of viral infection in cancer was established towards the beginning of 20th century. The study of tumour viruses, their oncogenes and different mechanisms employed by these viruses to subvert the growth-suppressive and pro-apoptotic functions of host tumour suppressor genes has laid the foundation of cancer biology. The human tumour viruses induce malignancies after a prolonged latency and in conjunction with other environmental and host factors. The eight known human tumour viruses contribute to nearly 10–15% of the cancers worldwide. Advancements in research on virus-related cancers offer a plethora of opportunities to fight cancer by preventing viral spread through vaccination and use of antivirals. Besides, recent developments on viral oncogenic mechanisms should allow development of novel and targeted approaches for control and treatment of virus-associated human cancers.
In this technologically savvy world, we witness mushrooming of sophisticated lines of softwares t... more In this technologically savvy world, we witness mushrooming of sophisticated lines of softwares that are extensively used for predicting epitopes towards developing vaccines and therapeutics. However, most softwares also generate bulky junk data along with data of interest. Here we devised a combined usage of B cell epitope prediction softwares - ABCpred along with additional online tools such as CLUSTAL W and Sequence Manipulation Suite for stringent selection of B cell epitopes. Dengue was used as model disease since the present diagnostic kits give false positives owing to cross-reactivity with other flaviviruses. The in silico predicted 240 unique B cell epitopes of dengue viruses were reduced to 87 serotype-specific epitopes using combination of softwares. Three best ranking epitopes of each dengue serotype were functionally validated by cloning corresponding DNA sequences in the pCBP vector and expressing these epitopes as calcium-binding protein fusion proteins. Western blot analysis and indirect ELISAs of purified fusion proteins confirmed that nearly fifty per cent of the epitopes were in close agreement with the predictions. Thus, stringent selection of epitopes using a combination of prediction softwares is likely to identify a compact library of B cell epitopes useful for developing more specific diagnostics in future.
Nucleolar assembly begins at the early G1 phase of cell cycle and is a hub of ribosomal DNA trans... more Nucleolar assembly begins at the early G1 phase of cell cycle and is a hub of ribosomal DNA transcription and ribosomal RNA (rRNA) biosynthesis. The newly formed rRNAs together with ribosomal proteins constitute the building block of the ribosomal machinery. Although ribosomal proteins play a major role in protein biosynthesis, their own regulation and expression is rather poorly understood. Here, we investigated the regulation ribosomal protein genes RPS27a, RPS24, RPS6, RPL9 and RPL4 in synchronized mammalian cell culture. The real-time quantitative PCR analysis indicated their expression during the mid to late G1 phase whereas the ribosomal RNA genes were expressed during the early G1 phase of cell cycle. The promoter reporter analysis of the RPS27a gene revealed that it could be synergistically stimulated by the transcription factors Sp1 and CREB. However, E2F1 appeared to negatively regulate the gene expression. The chromatin immunoprecipitation studies confirmed the promoter occupancy of Sp1, CREB and E2F1. While Sp1 and CREB binding enhanced the promoter occupancy of histone acetyltransferases PCAF, p300 and CBP, E2F1 facilitated the recruitment of histone deacetylases. Both acetylation (H4ac, H3K14ac) and methylation (H3K9me3) marks were observed in the RPS27a promoter region suggesting their important regulatory role in gene expression. Since the promoter regions of most ribosomal protein genes are well conserved, we propose their orchestrated regulation and synthesis during the cell cycle facilitates ribosome biogenesis.
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Papers by Vijay Kumar
ubiquitin ligases are tightly regulated both at transcriptional and post-translational levels. The E3 ubiquitin ligase TRUSS (tumor necrosis factor receptor-associated ubiquitous scaffolding and signaling protein) which negatively regulates c-Myc, are found down-regulated in most human cancer cell lines. However, the mechanism of regulation of intracellular levels of TRUSS remains elusive. Here we show that TRUSS is expressed majorly during the G1 phase of cell cycle and its level starts to decline with the expression of S-phase specific E3 ligase Skp2. Enforced expression of Skp2 led to a marked increase in the ubiquitination of TRUSS after its phosphorylation by GSK3β and followed by rapid proteolytic degradation. Our coimmunoprecipitation studies suggested a direct interaction between Skp2 and TRUSS through the LRR motif of Skp2. Interestingly, the human tumor samples that exhibited elevated expression of Skp2, showed relatively poor expression of TRUSS. Further, enforced expression of HBx, the oncoprotein of Hepatitis B virus which is known to stabilize c-Myc and enhance its oncogenic potential, led to the intracellular accumulation of TRUSS as well as c-Myc. Apparently, HBx also interacted with TRUSS which negatively impacted the TRUSS-c-Myc and TRUSS-Skp2 interactions leading to stabilization of TRUSS. Thus, the present study suggests that TRUSS is a novel substrate of E3 ligase Skp2 and that disruption of TRUSS-Skp2
interaction by viral oncoproteins could lead to pathophysiological sequelae.
Though Yukyung Karne has been reported to be clinically effective, the molecular mechanism of its anti-metstatic
action remains elusive.
Methods: The cytotoxic property of Yukyung Karne was evaluated by crystal violet staining while its ability to induce
ceramide production was analyzed by sphingomyelinase assay. The anti-metastatic property was investigated using
adhesion, invasion, migration and colony formation assays. The effect of Yukyung Karne on the expression of extracellular
matrix components, and epithelial and mesenchymal markers were evaluated by confocal microscopy and
western blotting.
Results: Yukyung Karne exhibited a strong anti-metastatic property by significantly reducing the invasion,
migration and colony formation ability of ovarian cancer cells. Besides it inhibited the levels of biomarkers
involved in epithelial to mesenchymal transition such as down-regulation of vimentin and N-cadherin and
up-regulation of epithelial E-cadherin. Yukyung Karne also induced the neutral sphingomyelinase II (nSMNaseII)
enzyme activity that is known to hydrolyze sphingomyelins into pro-apoptotic intracellular molecule ceramide.
Conclusions: The study provides some compelling evidences supporting the anti-metastatic potential of Yukyung
Karne which strongly suggests its possible usage as a promising alternative medicine. Thus, Yukyung Karne may
be used as an anticance
and several host proteins that are known to support the development and progression of hepatocellular
carcinoma (HCC). While overexpression of the nucleolar phosphoprotein, nucleophosmin (NPM), correlates
with HCC progression, its upregulation by viral HBx and the resulting impact on perturbed nucleolar functions
remain enigmatic. The present study shows that HBx up-regulates NPM levels and hijacks its functions to
promote cellular proliferation. We found that HBx expression stabilizes NPM through post-translational modifications.
Enhanced CDK2-mediated phosphorylation of NPM at Thr199 upon HBx expression prevented its proteolytic cleavage and provided resistance to apoptosis. Further, HBx directly interacted with the C-terminal domain of NPM and got translocated into the nucleolus where it facilitated the recruitment of RNA polymerase I transcriptional machinery onto the rDNA promoter. Our results indicate that HBx enhances rDNA transcription via a novel regulatory mechanism involving acetylation of NPM and the subsequent depletion of histones from the rDNA promoter. Enhanced production of ribosomal RNA resulting from co-expression of HBx and NPM
promoted ribosome biogenesis, cellular proliferation and transformation. Taken together, our study strongly suggests an important role of NPM in mediating the oncogenic effects of HBx and the corresponding nucleolar perturbations induced by this viral oncoprotein.
Methods: The UBF gene expression was investigated after co-expressing HBx in immortalized human hepatocytes (IHH) and human hepatoma Huh7 cells. Gene expression analysis involved estimation of mRNA level by real-time PCR, western blotting of protein, chromatin immune-precipitation assay, BrdU incorporation assay and soft agar colony formation assay. UBF expression was also investigated in an HBx transgenic mouse model of HCC to get a better mechanistic insight under more physiological conditions.
Results: Ectopic expression of HBx in IHH as well as Huh7 cells led to a marked increase in UBF expression both at mRNA and protein levels. Elevated levels of UBF were also observed in the hepatic tumors of HBx transgenic mice. Our ChIP studies revealed a marked increase in the occupancy of c-Myc on the UBF gene promoter in the presence of HBx and increase in its transcription. Enhanced UBF expression under the HBx microenvironment led to a marked increase in cell proliferation and transformation of IHH cells.
Conclusions: Our study provides some compelling evidences in support of HBx-mediated increase in UBF levels that abets oncogenic onslaught in hepatic cells by increasing rDNA transcription and ribosome biogenesis.
cancer. Although the incidence rates remain higher in more
developed countries, mortality and morbidity are higher in less
developed regions, attributable to late detection and lack of access to advanced medical amenities. Regardless of epidemiological prevalence, the etiological determinants of breast cancer development remain common and include age, family history, genetic risk factors such as BRCA1 and BRCA2 mutations and hormonal risk factors. Although viruses have been etiologically associated with many cancers,
a viral etiology to breast cancer is at best speculative. There is
conflicting evidence of roles of viruses such as human mammary tumor virus, human papillomavirus, Epstein-Barr virus, human cytomegalovirus, herpes simplex virus and measles virus in breast cancer development. Nonetheless, a recent development that levels of mutagenic antiviral enzyme APOBEC3B are elevated in a majority of breast cancers warrants a re-visit to the role of viruses in breast cancer pathogenesis. Keeping in view the current scenario, there is
an urgency to assess the worldwide trends and risk factors for
prediction of future scenarios with the ultimate goal of developing effective, affordable and prioritized approaches for breast cancer control.
Methods: The growth inhibition property of YK was evaluated in SKOV6, IHH, HepG2 and HEK293 cell lines using MTT assay. The pro-apoptotic activity of drug was analyzed by terminal deoxynuleotidyl transferase dUTP nick end labeling (TUNEL) and DNA fragmentation assays. Confocal microscopy was used to show the release of cytochrome c and its co-localization with mitochondria with the help of dsRed mitotracker in SKOV6 cells. The inhibition in cell proliferation was also visualized by confocal microscopy after BrDU incorporation. The activation of tumor suppressor p53 was evaluated by Western blotting while the VEGF level in culture supernatant was measured by a colorimetric method.
Results: YK specifically and efficiently induced apoptotic killing of the human ovarian cancer SKOV6 cells as indicated by increased DNA fragmentation and nick end DNA labeling. Confocal microscopy suggested inhibition of cell proliferation and increase in cytochrome c release via perturbation in mitochondrial membrane potential (m). Further, YK up-regulated the expression of tumor suppressor p53 and key cyclin-dependent kinase inhibitor p21, and inhibited VEGF secretion by cells. Interestingly, YK also exhibited a synergy with paclitaxel which a well-known anti-cancer therapeutic drug.
Conclusions: The pharmacological properties of YK to impose growth arrest and trigger pro-apoptotic death in cells amply justifies its usage in primary as well as adjunct therapy for ovarian cancer.
towards the beginning of 20th century. The study of
tumour viruses, their oncogenes and different mechanisms
employed by these viruses to subvert the
growth-suppressive and pro-apoptotic functions of
host tumour suppressor genes has laid the foundation
of cancer biology. The human tumour viruses induce
malignancies after a prolonged latency and in conjunction
with other environmental and host factors.
The eight known human tumour viruses contribute to
nearly 10–15% of the cancers worldwide. Advancements
in research on virus-related cancers offer a
plethora of opportunities to fight cancer by preventing
viral spread through vaccination and use of antivirals.
Besides, recent developments on viral oncogenic
mechanisms should allow development of novel and
targeted approaches for control and treatment of
virus-associated human cancers.
ubiquitin ligases are tightly regulated both at transcriptional and post-translational levels. The E3 ubiquitin ligase TRUSS (tumor necrosis factor receptor-associated ubiquitous scaffolding and signaling protein) which negatively regulates c-Myc, are found down-regulated in most human cancer cell lines. However, the mechanism of regulation of intracellular levels of TRUSS remains elusive. Here we show that TRUSS is expressed majorly during the G1 phase of cell cycle and its level starts to decline with the expression of S-phase specific E3 ligase Skp2. Enforced expression of Skp2 led to a marked increase in the ubiquitination of TRUSS after its phosphorylation by GSK3β and followed by rapid proteolytic degradation. Our coimmunoprecipitation studies suggested a direct interaction between Skp2 and TRUSS through the LRR motif of Skp2. Interestingly, the human tumor samples that exhibited elevated expression of Skp2, showed relatively poor expression of TRUSS. Further, enforced expression of HBx, the oncoprotein of Hepatitis B virus which is known to stabilize c-Myc and enhance its oncogenic potential, led to the intracellular accumulation of TRUSS as well as c-Myc. Apparently, HBx also interacted with TRUSS which negatively impacted the TRUSS-c-Myc and TRUSS-Skp2 interactions leading to stabilization of TRUSS. Thus, the present study suggests that TRUSS is a novel substrate of E3 ligase Skp2 and that disruption of TRUSS-Skp2
interaction by viral oncoproteins could lead to pathophysiological sequelae.
Though Yukyung Karne has been reported to be clinically effective, the molecular mechanism of its anti-metstatic
action remains elusive.
Methods: The cytotoxic property of Yukyung Karne was evaluated by crystal violet staining while its ability to induce
ceramide production was analyzed by sphingomyelinase assay. The anti-metastatic property was investigated using
adhesion, invasion, migration and colony formation assays. The effect of Yukyung Karne on the expression of extracellular
matrix components, and epithelial and mesenchymal markers were evaluated by confocal microscopy and
western blotting.
Results: Yukyung Karne exhibited a strong anti-metastatic property by significantly reducing the invasion,
migration and colony formation ability of ovarian cancer cells. Besides it inhibited the levels of biomarkers
involved in epithelial to mesenchymal transition such as down-regulation of vimentin and N-cadherin and
up-regulation of epithelial E-cadherin. Yukyung Karne also induced the neutral sphingomyelinase II (nSMNaseII)
enzyme activity that is known to hydrolyze sphingomyelins into pro-apoptotic intracellular molecule ceramide.
Conclusions: The study provides some compelling evidences supporting the anti-metastatic potential of Yukyung
Karne which strongly suggests its possible usage as a promising alternative medicine. Thus, Yukyung Karne may
be used as an anticance
and several host proteins that are known to support the development and progression of hepatocellular
carcinoma (HCC). While overexpression of the nucleolar phosphoprotein, nucleophosmin (NPM), correlates
with HCC progression, its upregulation by viral HBx and the resulting impact on perturbed nucleolar functions
remain enigmatic. The present study shows that HBx up-regulates NPM levels and hijacks its functions to
promote cellular proliferation. We found that HBx expression stabilizes NPM through post-translational modifications.
Enhanced CDK2-mediated phosphorylation of NPM at Thr199 upon HBx expression prevented its proteolytic cleavage and provided resistance to apoptosis. Further, HBx directly interacted with the C-terminal domain of NPM and got translocated into the nucleolus where it facilitated the recruitment of RNA polymerase I transcriptional machinery onto the rDNA promoter. Our results indicate that HBx enhances rDNA transcription via a novel regulatory mechanism involving acetylation of NPM and the subsequent depletion of histones from the rDNA promoter. Enhanced production of ribosomal RNA resulting from co-expression of HBx and NPM
promoted ribosome biogenesis, cellular proliferation and transformation. Taken together, our study strongly suggests an important role of NPM in mediating the oncogenic effects of HBx and the corresponding nucleolar perturbations induced by this viral oncoprotein.
Methods: The UBF gene expression was investigated after co-expressing HBx in immortalized human hepatocytes (IHH) and human hepatoma Huh7 cells. Gene expression analysis involved estimation of mRNA level by real-time PCR, western blotting of protein, chromatin immune-precipitation assay, BrdU incorporation assay and soft agar colony formation assay. UBF expression was also investigated in an HBx transgenic mouse model of HCC to get a better mechanistic insight under more physiological conditions.
Results: Ectopic expression of HBx in IHH as well as Huh7 cells led to a marked increase in UBF expression both at mRNA and protein levels. Elevated levels of UBF were also observed in the hepatic tumors of HBx transgenic mice. Our ChIP studies revealed a marked increase in the occupancy of c-Myc on the UBF gene promoter in the presence of HBx and increase in its transcription. Enhanced UBF expression under the HBx microenvironment led to a marked increase in cell proliferation and transformation of IHH cells.
Conclusions: Our study provides some compelling evidences in support of HBx-mediated increase in UBF levels that abets oncogenic onslaught in hepatic cells by increasing rDNA transcription and ribosome biogenesis.
cancer. Although the incidence rates remain higher in more
developed countries, mortality and morbidity are higher in less
developed regions, attributable to late detection and lack of access to advanced medical amenities. Regardless of epidemiological prevalence, the etiological determinants of breast cancer development remain common and include age, family history, genetic risk factors such as BRCA1 and BRCA2 mutations and hormonal risk factors. Although viruses have been etiologically associated with many cancers,
a viral etiology to breast cancer is at best speculative. There is
conflicting evidence of roles of viruses such as human mammary tumor virus, human papillomavirus, Epstein-Barr virus, human cytomegalovirus, herpes simplex virus and measles virus in breast cancer development. Nonetheless, a recent development that levels of mutagenic antiviral enzyme APOBEC3B are elevated in a majority of breast cancers warrants a re-visit to the role of viruses in breast cancer pathogenesis. Keeping in view the current scenario, there is
an urgency to assess the worldwide trends and risk factors for
prediction of future scenarios with the ultimate goal of developing effective, affordable and prioritized approaches for breast cancer control.
Methods: The growth inhibition property of YK was evaluated in SKOV6, IHH, HepG2 and HEK293 cell lines using MTT assay. The pro-apoptotic activity of drug was analyzed by terminal deoxynuleotidyl transferase dUTP nick end labeling (TUNEL) and DNA fragmentation assays. Confocal microscopy was used to show the release of cytochrome c and its co-localization with mitochondria with the help of dsRed mitotracker in SKOV6 cells. The inhibition in cell proliferation was also visualized by confocal microscopy after BrDU incorporation. The activation of tumor suppressor p53 was evaluated by Western blotting while the VEGF level in culture supernatant was measured by a colorimetric method.
Results: YK specifically and efficiently induced apoptotic killing of the human ovarian cancer SKOV6 cells as indicated by increased DNA fragmentation and nick end DNA labeling. Confocal microscopy suggested inhibition of cell proliferation and increase in cytochrome c release via perturbation in mitochondrial membrane potential (m). Further, YK up-regulated the expression of tumor suppressor p53 and key cyclin-dependent kinase inhibitor p21, and inhibited VEGF secretion by cells. Interestingly, YK also exhibited a synergy with paclitaxel which a well-known anti-cancer therapeutic drug.
Conclusions: The pharmacological properties of YK to impose growth arrest and trigger pro-apoptotic death in cells amply justifies its usage in primary as well as adjunct therapy for ovarian cancer.
towards the beginning of 20th century. The study of
tumour viruses, their oncogenes and different mechanisms
employed by these viruses to subvert the
growth-suppressive and pro-apoptotic functions of
host tumour suppressor genes has laid the foundation
of cancer biology. The human tumour viruses induce
malignancies after a prolonged latency and in conjunction
with other environmental and host factors.
The eight known human tumour viruses contribute to
nearly 10–15% of the cancers worldwide. Advancements
in research on virus-related cancers offer a
plethora of opportunities to fight cancer by preventing
viral spread through vaccination and use of antivirals.
Besides, recent developments on viral oncogenic
mechanisms should allow development of novel and
targeted approaches for control and treatment of
virus-associated human cancers.