Jitendra Kumar Kanaujiya

Synthesis and cytotoxicity evaluation of (tetrahydro-β-carboline)-1,3,5-triazine hybrids as anticancer agents

A series of tetrahydro-beta-carbolines and 1,3,5-triazine hybrids have been synthesized and evaluated for their cytotoxicity against a panel of eight human cancer cell lines and normal human fibroblasts (NIH3T3). It led us to discovery of racemic compounds 69, 71 and 75, which are selectively cytotoxic towards KB (oral cancer) cell line with IC50 values of 105.8, 664.7 and 122.2 nM, respectively; while their enantiopure forms are less active and not selective. Enantiopure compound 42 showed 2.5 times more selectivity towards MCF7 cells over normal fibroblast NIH3T3 cells with an IC50 value of 740 nM, also arrests cell cycle in G1 phase and induces apoptosis in MCF7 and MDA MB231 cell lines.

2‐D gel electrophoresis‐based proteomic analysis reveals that ormeloxifen induces G0–G1 growth arrest and ERK‐mediated apoptosis in chronic myeloid leukemia cells K562

Ormeloxifen is a nonsteroidal selective estrogen receptor modulator (SERM) and has been shown to possess anticancer activities in breast and uterine cancer. Here, we show that ormeloxifen induces apoptosis in dose‐dependent manner in a variety of leukemia cells, more strikingly in K562. 2‐DE‐gel electrophoresis of K562 cells induced with ormeloxifen showed that 57 and 30% of proteins belong to apoptosis and cell‐cycle pathways, respectively. Our data demonstrate that ormeloxifen‐induced apoptosis in K562 cells involves activation of extracellular signal‐regulated kinases (ERKs) and subsequent cytochrome c release, leading to mitochondria‐mediated caspase‐3 activation. Ormeloxifen‐induced apoptosis via ERK activation was drastically inhibited by prior treatment of K562 cells with ERK inhibitor PD98059. Ormeloxifen also inhibits proliferation of K562 cells by blocking them in G0–G1 phase by inhibiting c‐myc promoter via ormeloxifen‐induced MBP‐1 (c‐myc promoter‐binding protein) and upregulation of p21 expression. We further show that ormeloxifen‐induced apoptosis in K562 is translatable to mononuclear cells isolated from chronic myeloid leukemia (CML) patients. Thus, ormeloxifen induces apoptosis in K562 cells via phosphorylation of ERK and arrests them in G0–G1 phase by reciprocal regulation of p21 and c‐myc. Therefore, inclusion of ormeloxifen in the therapy of chronic myeloid leukemia can be of potential utility.

E3 ubiquitin ligase Fbw7 negatively regulates granulocytic differentiation by targeting G-CSFR for degradation

Tight control between activation and attenuation of granulocyte colony stimulating factor receptor (G-CSFR) signaling is essential to regulate survival, proliferation and differentiation of myeloid progenitor cells. Previous studies demonstrated negative regulation of G-CSFR through endosomal-lysosomal routing and ubiquitin-proteasome mediated degradation. However, very few E3 ubiquitin ligases are known to target G-CSFR for ubiquitin-proteasome pathway. Here we identified F-box and WD repeat domain-containing 7 (Fbw7), a substrate recognizing component of Skp-Cullin-F box (SCF) E3 ubiquitin Ligase physically associates with G-CSFR and promotes its ubiquitin-mediated proteasomal degradation. Our data shows that Fbw7 also interacts with and degrades G-CSFR-T718 (a truncated mutant of G-CSFR found in severe congenital neutropenia/acute myeloid leukemia (SCN/AML patients)) though at a quite slower rate compared to G-CSFR. We further show that glycogen synthase kinase 3 beta (GSK3β), like Fbw7 also targets G-CSFR and G-CSFR-T718 for degradation; however, Fbw7 and GSK3β are interdependent in targeting G-CSFR/G-CSFR-T718 for degradation because they are unable to degrade G-CSFR individually when either of them is knocked down. We further show that Fbw7 mediated downregulation of G-CSFR inhibits signal transducer and activator of transcription 3 (STAT3) phosphorylation which is required for G-CSF dependent granulocytic differentiation. In addition, our data also shows that inhibition of Fbw7 restores G-CSFR signaling leading to enhanced STAT3 activity resulting in massive granulocytic differentiation. These data indicate that Fbw7 together with GSK3β negatively regulates G-CSFR expression and its downstream signaling.

Proteomic identification of E6AP as a molecular target of tamoxifen in MCF7 cells.

Tamoxifen (Tam) is most widely used selective estrogen receptor modulator (SERM) for treatment of hormone‐responsive breast cancer. Despite being regularly used in clinical therapy for breast cancer since 1971, the mechanism of Tam action remains largely unclear. In order to gain insights into Tam‐mediated antibreast cancer actions, we applied 2DE and MS based proteomics approach to identify target proteins of Tam. We identified E6‐associated protein, i.e. E6AP (UBE3A) among others to be regulated by Tam that otherwise is upregulated in breast tumors. We confirmed our 2DE finding by immunoblotting and further show that Tam leads to inhibition of E6AP expression presumably by promoting its autoubiquitination, which is coupled with nuclear export and subsequent proteasome‐mediated degradation. Furthermore, we show that Tam‐ and siE6AP‐mediated inhibition of E6AP leads to enhanced G0–G1 growth arrest and apoptosis, which is also evident from significant upregulation of cytochrome‐c, Bax, p21, and PARP cleavage. Taken together, our data suggest that, Tam‐targeted E6AP inhibition is in fact required for Tam‐mediated antibreast cancer actions. Thus, E6AP may be a therapeutic target in breast cancer.

E3 ubiquitin ligase E6AP negatively regulates adipogenesis by downregulating proadipogenic factor C/EBPalpha.

CCAAT/Enhancer Binding Protein Alpha (C/EBPα) is a key transcription factor involved in the adipocyte differentiation. Here for the first time we demonstrate that E6AP, an E3 ubiquitin ligase inhibits adipocyte differentiation in 3T3-L1 cells as revealed by reduced lipid staining with oil red. Knock down of E6AP in mouse 3T3L1 preadipocytes is sufficient to convert them to adipocytes independent of external hormonal induction. C/EBPα protein level is drastically increased in E6AP deficient 3T3L1 preadipocytes while inverse is observed when wild type E6AP is over expressed. We show that transient transfection of wild type E6AP downregulates C/EBPα protein expression in a dose dependent manner while catalytically inactive E6AP-C843A rather stabilizes it. In addition, wild type E6AP inhibits expression of proadipogenic genes while E6AP-C843A enhances them. More importantly, overexpression of E6AP-C843A in mesenchymal progenitor cells promotes accumulation of lipid droplets while there is drastically reduced lipid droplet formation when E6AP is over expressed. Taken together, our finding suggests that E6AP may negatively control adipogenesis by inhibiting C/EBPα expression by targeting it to ubiquitin-proteasome pathway for degradation.

E6AP, an E3 ubiquitin ligase negatively regulates granulopoiesis by targeting transcription factor C/EBPα for ubiquitin-mediated proteasome degradation.

CCAAT/enhancer-binding protein alpha (C/EBPα) is an important transcription factor involved in granulocytic differentiation. Here, for the first time we demonstrate that E6-associated protein (E6AP), an E3 ubiquitin ligase targets C/EBPα for ubiquitin-mediated proteasome degradation and thereby negatively modulates its functions. Wild-type E6AP promotes ubiquitin dependent proteasome degradation of C/EBPα, while catalytically inactive E6-associated protein having cysteine replaced with alanine at amino-acid position 843 (E6AP-C843A) rather stabilizes it. Further, these two proteins physically associate both in non-myeloid (overexpressed human embryonic kidney epithelium) and myeloid cells. We show that E6AP-mediated degradation of C/EBPα protein expression curtails its transactivation potential on its target genes. Noticeably, E6AP degrades both wild-type 42 kDa CCAAT-enhancer-binding protein alpha (p42C/EBPα) and mutant isoform 30 kDa CCAAT-enhancer-binding protein alpha (p30C/EBPα), this may explain perturbed p42C/EBPα/p30C/EBPα ratio often observed in acute myeloid leukemia (AML). We show that overexpression of catalytically inactive E6AP-C843A in C/EBPα inducible K562- p42C/EBPα-estrogen receptor (ER) cells inhibits β-estradiol (E2)-induced C/EBPα degradation leading to enhanced granulocytic differentiation. This enhanced granulocytic differentiation upon E2-induced activation of C/EBPα in C/EBPα stably transfected cells (β-estradiol inducible K562 cells stably expressing p42C/EBPα-ER (K562-C/EBPα-p42-ER)) was further substantiated by siE6AP-mediated knockdown of E6AP in both K562-C/EBPα-p42-ER and 32dcl3 (32D clone 3, a cell line widely used model for in vitro study of hematopoietic cell proliferation, differentiation, and apoptosis) cells. Taken together, our data suggest that E6AP targeted C/EBPα protein degradation may provide a possible explanation for both loss of expression and/or functional inactivation of C/EBPα often experienced in myeloid leukemia.

E3 Ubiquitin Ligase Fbw7 Negatively Regulates Osteoblast Differentiation by Targeting Runx2 for Degradation.

Runx2, a master regulator of osteoblast differentiation, is tightly regulated at both transcriptional and post-translational levels. Post-translational modifications such as phosphorylation and ubiquitination have differential effects on Runx2 functions. Here, we show that the reduced expression and functions of Runx2 upon its phosphorylation by GSK3β are mediated by its ubiquitin-mediated degradation through E3 ubiquitin ligase Fbw7α. Fbw7α through its WD domain interacts with Runx2 both in a heterologous (HEK293T cells) system as well as in osteoblasts. GSK3β was also present in the same complex as determined by co-immunoprecipitation. Furthermore, overexpression of either Fbw7α or GSK3β was sufficient to down-regulate endogenous Runx2 expression and function; however, both failed to inhibit endogenous Runx2 when either of them was depleted in osteoblasts. Fbw7α-mediated inhibition of Runx2 expression also led to reduced Runx2 transactivation and osteoblast differentiation. In contrast, inhibition of Fbw7α restored Runx2 levels and promoted osteoblast differentiation. We also observed reciprocal expression levels of Runx2 and Fbw7α in models of bone loss such as lactating (physiological bone loss condition) and ovariectomized (induction of surgical menopause) animals that show reduced Runx2 and enhanced Fbw7α, whereas this was reversed in the estrogen-treated ovariectomized animals. In addition, methylprednisolone (a synthetic glucocorticoid) treatment to neonatal rats showed a temporal decrease in Runx2 with a reciprocal increase in Fbw7 in their calvarium. Taken together, these data demonstrate that Fbw7α negatively regulates osteogenesis by targeting Runx2 for ubiquitin-mediated degradation in a GSK3β-dependent manner and thus provides a plausible explanation for GSK3β-mediated bone loss as described before.

Proteomic identification of Profilin1 as a corepressor of estrogen receptor alpha in MCF7 breast cancer cells

Nuclear receptor coregulators play an important role in the transcriptional regulation of nuclear receptors. In the present study, we aimed to identify estrogen receptor α (ERα) interacting proteins in Tamoxifen treated MCF7 cells. Using in vitro GST‐pull down assay with ERα ligand‐binding domain (ERα‐LBD) and MS‐based proteomics approach we identified Profilin1 as a novel ERα interacting protein. Profilin1 contains I/LXX/L/H/I amino acid signature motif required for corepressor interaction with ERα. We show that these two proteins physically interact with each other both in vitro as well as in vivo by GST‐pull down and coimmunoprecipitation, respectively. We further show that these two proteins also colocalize together in the nucleus. Previous studies have reported reduced expression of Profilin1 in breast cancer; and here we found that Tamoxifen increases Profilin1 expression in MCF7 cells. Our data demonstrate that over expression of Profilin1 inhibits ERα‐mediated transcriptional activation as well as its downstream target genes in ERα positive breast cancer cells MCF7. In addition, Profilin1 overexpression in MCF7 cells leads to inhibition of cell proliferation that apparently is due to enhanced apoptosis. In nutshell, these data indicate that MS‐based proteomics approach identifies a novel ERα interacting protein Profilin1 that serves as a putative corepressor of ERα functions.

Ectopic expression of hC/EBPs in breast tumor cells induces apoptosis

CCAAT/enhancer binding proteins (C/EBPs) are a group of transcription factors which have been implicated in cellular proliferation, terminal differentiation, and apoptosis in a variety of tissues including mammary gland. Owing to its role in various cellular functions, inactivation of C/EBP proteins is central to the pathogenesis of many disorders. Recent reports suggest that expression as well as function of C/EBP proteins is deregulated in breast tumors. Although, role of C/EBPs in growth arrest in mammary tissues has been studied in much detail; their role in apoptosis is relatively less explored. In the present study, we have assessed if breast tumors evade apoptosis and grow faster by down regulating and inhibiting the C/EBP proteins, C/EBPα in particular. Our data shows that ectopic expression of human C/EBPs in breast tumor cells inhibits proliferation and induces apoptosis which is likely to be associated with caspase dependent pathway.

Proteomic approaches in myeloid leukemia

After human genome is decoded, the characterization of the proteins is the next challenging task. The study of the complete protein complement of the genome, the ‘proteome’ referred to as proteomics, is an important tool for the identification of new therapeutic targets. Research efforts are underway to develop the technology necessary to compare the specific protein profiles of diseased versus healthy states. These technologies provide a wealth of information by rapidly generating large quantities of data. These data can be useful for predictive mathematical descriptions of biological systems for rapid identification of novel therapeutic targets and identification of biomarkers in metabolic disorders. In recent years, using proteomics, we and others have identified various interacting as well as target proteins, PTMs and protein markers in myeloid leukemia. This review summarizes the usage of proteomics in recent years as an important technique in defining the proteome of myeloid leukemia, which has helped in elaborate understanding of the disease and has provided new avenues for developing better therapeutics.