Exploring Anticancer Properties of Medicinal Plants against Breast Cancer by Downregulating Human Epidermal Growth Factor Receptor 2.

Plants have a history of being employed in managing breast cancer. However, no scientific evidence supports the idea that these plants can effectively reduce the level of HER2 expression. In this study, extracts from 10 medicinal plants were evaluated for their anticancer properties against HER2-positive breast cancer cells through various methods, including the SRB assay, comet assay, annexin V-FITC dual staining, and immunoblotting. All extracts exerted antiproliferative activity against HER2-positive breast cancer cells. Furthermore, Terminalia chebula (T. chebula), Berberis aristata (B. aristata), and Mucuna pruriens (M. pruriens) reduced HER2 expression in tested cell lines. In addition, an increased Bax/Bcl-2 ratio was observed after the treatment. A comparative proteomics study showed modulation in the proteome profile of breast cancer cells after treatment with T. chebula, B. aristata, Punica granatum, M. pruriens, and Acorus calamus. Metabolic profiling of lead plants revealed the existence of multiple anticancer compounds. Our study demonstrates the considerable potential of the mentioned plants as innovative therapies for HER2-positive breast cancer.

Secreted mutant calreticulins as rogue cytokines in myeloproliferative neoplasms.

Mutant calreticulin (CALR) proteins resulting from a -1/+2 frameshifting mutation of the CALR exon 9 carry a novel C-terminal amino acid sequence and drive the development of myeloproliferative neoplasms (MPNs). Mutant CALRs were shown to interact with and activate the thrombopoietin receptor (TpoR/MPL) in the same cell. We report that mutant CALR proteins are secreted and can be found in patient plasma at levels up to 160 ng/mL, with a mean of 25.64 ng/mL. Plasma mutant CALR is found in complex with soluble transferrin receptor 1 (sTFR1) that acts as a carrier protein and increases mutant CALR half-life. Recombinant mutant CALR proteins bound and activated the TpoR in cell lines and primary megakaryocytic progenitors from patients with mutated CALR in which they drive thrombopoietin-independent colony formation. Importantly, the CALR-sTFR1 complex remains functional for TpoR activation. By bioluminescence resonance energy transfer assay, we show that mutant CALR proteins produced in 1 cell can specifically interact in trans with the TpoR on a target cell. In comparison with cells that only carry TpoR, cells that carry both TpoR and mutant CALR are hypersensitive to exogenous mutant CALR proteins and respond to levels of mutant CALR proteins similar to those in patient plasma. This is consistent with CALR-mutated cells that expose TpoR carrying immature N-linked sugars at the cell surface. Thus, secreted mutant CALR proteins will act more specifically on the MPN clone. In conclusion, a chaperone, CALR, can turn into a rogue cytokine through somatic mutation of its encoding gene.

In and out: Traffic and dynamics of thrombopoietin receptor.

Thrombopoiesis had long been a challenging area of study due to the rarity of megakaryocyte precursors in the bone marrow and the incomplete understanding of its regulatory cytokines. A breakthrough was achieved in the early 1990s with the discovery of the thrombopoietin receptor (TpoR) and its ligand thrombopoietin (TPO). This accelerated research in thrombopoiesis, including the uncovering of the molecular basis of myeloproliferative neoplasms (MPN) and the advent of drugs to treat thrombocytopenic purpura. TpoR mutations affecting its membrane dynamics or transport were increasingly associated with pathologies such as MPN and thrombocytosis. It also became apparent that TpoR affected hematopoietic stem cell (HSC) quiescence while priming hematopoietic stem cells (HSCs) towards the megakaryocyte lineage. Thorough knowledge of TpoR surface localization, dimerization, dynamics and stability is therefore crucial to understanding thrombopoiesis and related pathologies. In this review, we will discuss the mechanisms of TpoR traffic. We will focus on the recent progress in TpoR membrane dynamics and highlight the areas that remain unexplored.

DaiCee: A database for anti-cancer compounds with targets and side effect profiles.

Identification of the toxicity of compounds is more crucial before entering clinical trials. Awareness of physiochemical properties, possible targets and side effects has become a major public health issue to reduce risks. Experimental determination of analyzing the physiochemical properties of a drug, their interaction with specific receptors and identifying their side-effects remain challenging is time consuming and costly. We describe a manually compiled database named DaiCee database, which contains 2100 anticancer drugs with information on their physiochemical properties, targets of action and side effects. It includes both synthetic and herbal anti-cancer compounds. It allows the search for SMILES notation, Lipinski's and ADME/T properties, targets and side effect profiles of the drugs. This helps to identify drugs with effective anticancer properties, their toxic nature, drug-likeness for in-vitro and in-vivo experiments. It also used for comparative analysis and screening of effective anticancer drugs using available data for compounds in the database. The database will be updated regularly to provide the users with latest information. The database is available at the URL http://www.hccbif.org/usersearch.php.

Calreticulin mutants as oncogenic rogue chaperones for TpoR and traffic-defective pathogenic TpoR mutants.

Calreticulin (CALR) +1 frameshift mutations in exon 9 are prevalent in myeloproliferative neoplasms. Mutant CALRs possess a new C-terminal sequence rich in positively charged amino acids, leading to activation of the thrombopoietin receptor (TpoR/MPL). We show that the new sequence endows the mutant CALR with rogue chaperone activity, stabilizing a dimeric state and transporting TpoR and mutants thereof to the cell surface in states that would not pass quality control; this function is absolutely required for oncogenic transformation. Mutant CALRs determine traffic via the secretory pathway of partially immature TpoR, as they protect N117-linked glycans from further processing in the Golgi apparatus. A number of engineered or disease-associated TpoRs such as TpoR/MPL R102P, which causes congenital thrombocytopenia, are rescued for traffic and function by mutant CALRs, which can also overcome endoplasmic reticulum retention signals on TpoR. In addition to requiring N-glycosylation of TpoR, mutant CALRs require a hydrophobic patch located in the extracellular domain of TpoR to induce TpoR thermal stability and initial intracellular activation, whereas full activation requires cell surface localization of TpoR. Thus, mutant CALRs are rogue chaperones for TpoR and traffic-defective TpoR mutants, a function required for the oncogenic effects.

Octyl gallate and gallic acid isolated from Terminalia bellarica regulates normal cell cycle in human breast cancer cell lines.

Herbal medicines stand unique and effective in treating human diseases. Terminalia bellarica (T. bellarica) is a potent medicinal herb, with a wide range of pharmacological activities. The present study was aimed to evaluate the effect of octyl gallate (OG) and gallic acid (GA) isolated from methanolic fruit extract of T. bellirica to inhibit the survival of breast cancer cells (MCF-7 & MDA-MB-231). Both OG & GA exhibited decreased MCF-7 & MDA-MB-231 survival and induced apoptosis, with IC50 value of OG and GA as 40 µM and 80 µM respectively. No toxic effect was observed on normal breast cells (MCF-10A). The compounds inhibited cell cycle progression by altering the expression of the cell cycle regulators (Cyclin D1, D3, CDK-4, CDK-6, p18 INK4, p21Waf-1 and p27 KIP). Octyl gallate was more effective at low concentrations than GA. In-silico results provided stable interactions between the compounds and target proteins. The present investigation proved the downregulation of positive cell cycle regulators and upregulation of negative cell cycle regulators inducing apoptosis in compound-treated breast cancer cells. Hence, both the compounds may serve as potential anticancer agents and could be developed as breast cancer drugs, with further explorations.

Activity of nonmuscle myosin II isoforms determines localization at the cleavage furrow of megakaryocytes.

Megakaryocyte polyploidy is characterized by cytokinesis failure resulting from defects in contractile forces at the cleavage furrow. Although immature megakaryocytes express 2 nonmuscle myosin II isoforms (MYH9 [NMIIA] and MYH10 [NMIIB]), only NMIIB localizes at the cleavage furrow, and its subsequent absence contributes to polyploidy. In this study, we tried to understand why the abundant NMIIA does not localize at the furrow by focusing on the RhoA/ROCK pathway that has a low activity in polyploid megakaryocytes. We observed that under low RhoA activity, NMII isoforms presented different activity that determined their localization. Inhibition of RhoA/ROCK signaling abolished the localization of NMIIB, whereas constitutively active RhoA induced NMIIA at the cleavage furrow. Thus, although high RhoA activity favored the localization of both the isoforms, only NMIIB could localize at the furrow at low RhoA activity. This was further confirmed in erythroblasts that have a higher basal RhoA activity than megakaryocytes and express both NMIIA and NMIIB at the cleavage furrow. Decreased RhoA activity in erythroblasts abolished localization of NMIIA but not of NMIIB from the furrow. This differential localization was related to differences in actin turnover. Megakaryocytes had a higher actin turnover compared with erythroblasts. Strikingly, inhibition of actin polymerization was found to be sufficient to recapitulate the effects of inhibition of RhoA/ROCK pathway on NMII isoform localization; thus, cytokinesis failure in megakaryocytes is the consequence of both the absence of NMIIB and a low RhoA activity that impairs NMIIA localization at the cleavage furrow through increased actin turnover.

Uncoupling of the Hippo and Rho pathways allows megakaryocytes to escape the tetraploid checkpoint.

Megakaryocytes are naturally polyploid cells that increase their ploidy by endomitosis. However, very little is known regarding the mechanism by which they escape the tetraploid checkpoint to become polyploid. Recently, it has been shown that the tetraploid checkpoint was regulated by the Hippo-p53 pathway in response to a downregulation of Rho activity. We therefore analyzed the role of Hippo-p53 pathway in the regulation of human megakaryocyte polyploidy. Our results revealed that Hippo-p53 signaling pathway proteins are present and are functional in megakaryocytes. Although this pathway responds to the genotoxic stress agent etoposide, it is not activated in tetraploid or polyploid megakaryocytes. Furthermore, Hippo pathway was observed to be uncoupled from Rho activity. Additionally, polyploid megakaryocytes showed increased expression of YAP target genes when compared to diploid and tetraploid megakaryocytes. Although p53 knockdown increased both modal ploidy and proplatelet formation in megakaryocytes, YAP knockdown caused no significant change in ploidy while moderately affecting proplatelet formation. Interestingly, YAP knockdown reduced the mitochondrial mass in polyploid megakaryocytes and decreased expression of PGC1α, an important mitochondrial biogenesis regulator. Thus, the Hippo pathway is functional in megakaryocytes, but is not induced by tetraploidy. Additionally, YAP regulates the mitochondrial mass in polyploid megakaryocytes.

P53 activation inhibits all types of hematopoietic progenitors and all stages of megakaryopoiesis.

TP53 also known as p53 is a tumor suppressor gene mutated in a variety of cancers. P53 is involved in cell cycle, apoptosis and DNA repair mechanisms and is thus tightly controlled by many regulators. Recently, strategies to treat cancer have focused on the development of MDM2 antagonists to induce p53 stabilization and restore cell death in p53 non-mutated cancers. However, some of these molecules display adverse effects in patients including induction of thrombocytopenia. In the present study, we have explored the effect of SAR405838 not only on human megakaryopoiesis but also more generally on hematopoiesis. We compared its effect to MI-219 and Nutlin, which are less potent MDM2 antagonists than SAR405838. We found that all these compounds induce a deleterious effect on all types of hematopoietic progenitors, as well as on erythroid and megakaryocytic differentiation. Moreover, they inhibit both early and late stages of megakaryopoiesis including ploidization and proplatelet formation. In conclusion, MDM2 antagonists induced a major hematopoietic defect in vitro as well as an inhibition of all stages of megakaryopoiesis that may account for in vivo thrombocytopenia observed in treated patients.

Neonatal Persistent Exposure to 6-Propyl-2-thiouracil, a Thyroid-Disrupting Chemical, Differentially Modulates Expression of Hepatic Catalase and C/EBP-ß in Adult Rats.

Persistent exposure of rats to 6-propyl-2-thiouracil (PTU) from birth resulted in decreases in plasma thyroid hormone (TH) levels and hepatic expression of catalase and CCAAT enhancer binding protein ß (C/EBP-ß). Catalase promoter region (-185 to +52) that contains binding sites for C/EBP-ß showed an augmentation in the methylation level along with a change in methylation pattern of CpG islands in response to PTU treatment. PTU withdrawal on 30 days of birth restored TH levels and C/EBP-ß to control rats in adulthood. Although catalase expression was restored to some extent in adult rats in response to PTU withdrawal, a permanent change in its promoter CpG methylation pattern was recorded. The results suggest that downregulation of adult hepatic catalase gene in response to persistent neonatal PTU exposure may not solely be attributed to thyroid-disrupting properties of PTU. It is possible that besides thyroid-disrupting behavior, PTU may impair expression of hepatic catalase by altering methylation pattern of its promoter.

Cytotoxic Impact of Costunolide Isolated from Costus speciosus on Breast Cancer via Differential Regulation of Cell Cycle-An In-vitro and In-silico Approach.

Costunolide, a sesquiterpene lactone, is a biologically active molecule found in most of the medicinally valuable plants. The present study aims to evaluate the anticancer property of costunolide isolated from Costus speciosus against breast cancer cell lines (MCF-7 and MDA-MB-231). Costunolide effectively reduced the viability of both MCF-7 and MDA-MB-231 cell lines at an IC50 value of 40 µM. Flow cytometric analysis revealed costunolide mediated cell cycle arrest at G2/M phase in both the cell types. Western blotting results confirmed the alterations in the expression of cell cycle regulators (cyclin D1, D3, CDK-4, CDK-6, p18 INK4c, p21 CIP1/Waf-1 and p27 KIP1) and apoptosis inducers (caspase-3 and caspase-9) upon costunolide treatment in comparison with their expressions in normal breast cell line (MCF-10A). Costunolide mediated downregulation of positive cell cycle regulators and upregulation of negative cell cycle regulators were related to the induction of apoptosis in cancer cells. The above results were validated with in-silico results that predicted stable interactions between costunolide and cancer targets. Thus costunolide effectively induced breast cancer cell apoptosis targeting cell cycle regulation, and the compound can be used as an effective herbal therapeutic molecule to treat breast cancer with further explorations.

p27 and leukemia: cell cycle and beyond.

Cell division is the foundation to development and the regulation of cell cycle progression is therefore of paramount importance to the living organisms. Primary control of cell cycle is achieved by an array of cyclins and cyclin dependent kinases (CDKs). The functions of these cyclin-CDK complexes are again regulated by a host of cyclin dependent kinase inhibitors (CDKI). Till date CDKIs are broadly classified into two groups-INK4 family (p15, p16, p18, and p19) and the cip/kip family (p21, p27, and p57). Collectively these CDKIs regulate the progression from G1 to S phase of cell cycle. This review summarizes the functions of p27 while highlighting its emerging roles in leukemia.

Differential regulation of MCM7 and its intronic miRNA cluster miR-106b-25 during megakaryopoiesis induced polyploidy.

Megakaryocytes exit from mitotic cell cycle and enter a phase of repeated DNA replication without undergoing cell division, in a process termed as endomitosis of which little is known. We studied the expression of a DNA replication licensing factor mini chromosome maintenance protein 7 (MCM7) and its intronic miR-106b-25 cluster during mitotic and endo-mitotic cycles in megakaryocytic cell lines and in vitro cultured megakaryocytes obtained from human cord blood derived CD34(+) cells. Our results show that contrary to mitotic cell cycle, endomitosis proceeds with an un-coupling of the expression of MCM7 and miR-106b-25. This was attributed to the presence of a transcript variant of MCM7 which undergoes nonsense mediated decay (NMD). Additionally, miR-25 which was up regulated during endomitosis was found to promote megakaryopoiesis by inhibiting the expression of PTEN. Our study thus highlights the importance of a transcript variant of MCM7 destined for NMD in the modulation of megakaryopoiesis.

In vitro evaluation of anticancer potentials of lupeol isolated from Elephantopus scaber L. on MCF-7 cell line.

UNLABELLED: Lupeol is a triterpenoid, present in most of the medicinally effective plants and possess a wide range of biological activity against human diseases. The present study aims at evaluating the anticancer potentials of lupeol, isolated from the leaves of Elephantopus scaber L. and thereby explores its action on key cancer marker, Bcl-2. The effect of lupeol on the cell viability of MCF-7 was determined by MTT and lactate dehydrogenase assays at different concentrations. The efficacy of the compound to induce cell death was analyzed using AO/EtBr staining. Phase contrast microscopic analysis provided the changes in cell morphology of the compound treated normal breast cells (MCF-10A) and MCF-7 cells. The expression of Bcl-2 and Bcl-xL proteins in the normal, cancer and lupeol treated cancer cell was analyzed by western blotting. Lupeol induced an effective change in the cell viability of MCF-7 cells with IC50 concentration as 80 µM. Induction of cell death, change in cell morphology and population of the cancer cells was observed in the lupeol treated cells, but the normal cells were not affected. The compound effectively downregulated Bcl-2 and Bcl-xL protein expressions, which directly contribute for the induction of MCF-7 cell apoptosis. CONCLUSION: Thus, lupeol acts as an anticancer agent against MCF-7 cells and is a potent phytodrug to be explored further for its cytotoxic mechanism.

Carboxyl-terminal-dependent recruitment of nonmuscle myosin II to megakaryocyte contractile ring during polyploidization.

Endomitosis is a unique megakaryocyte (MK) differentiation process that is the consequence of a late cytokinesis failure associated with a contractile ring defect. Evidence from in vitro studies has revealed the distinct roles of 2 nonmuscle myosin IIs (NMIIs) on MK endomitosis: only NMII-B (MYH10), but not NMII-A (MYH9), is localized in the MK contractile ring and implicated in mitosis/endomitosis transition. Here, we studied 2 transgenic mouse models in which nonmuscle myosin heavy chain (NMHC) II-A was genetically replaced either by II-B or by a chimeric NMHCII that combined the head domain of II-A with the rod and tail domains of II-B. This study provides in vivo evidence on the specific role of NMII-B on MK polyploidization. It demonstrates that the carboxyl-terminal domain of the heavy chains determines myosin II localization to the MK contractile ring and is responsible for the specific role of NMII-B in MK polyploidization.

Supplementation of T3 recovers hypothyroid rat liver cells from oxidatively damaged inner mitochondrial membrane leading to apoptosis.

Hypothyroidism is a growing medical concern. There are conflicting reports regarding the mechanism of oxidative stress in hypothyroidism. Mitochondrial oxidative stress is pivotal to thyroid dysfunction. The present study aimed to delineate the effects of hepatic inner mitochondrial membrane dysfunction as a consequence of 6-n-propyl-2-thiouracil-induced hypothyroidism in rats. Increased oxidative stress predominance in the submitochondrial particles (SMP) and altered antioxidant defenses in the mitochondrial matrix fraction correlated with hepatocyte apoptosis. In order to check whether the effects caused by hypothyroidism are reversed by T3, the above parameters were evaluated in a subset of T3-treated hypothyroid rats. Complex I activity was inhibited in hypothyroid SMP, whereas T3 supplementation upregulated electron transport chain complexes. Higher mitochondrial H2O2 levels in hypothyroidism due to reduced matrix GPx activity culminated in severe oxidative damage to membrane lipids. SMP and matrix proteins were stabilised in hypothyroidism but exhibited increased carbonylation after T3 administration. Glutathione content was higher in both. Hepatocyte apoptosis was evident in hypothyroid liver sections; T3 administration, on the other hand, exerted antiapoptotic and proproliferative effects. Hence, thyroid hormone level critically regulates functional integrity of hepatic mitochondria; hypothyroidism injures mitochondrial membrane lipids leading to hepatocyte apoptosis, which is substantially recovered upon T3 supplementation.

In vitro and in silico evaluation of NF-κB targeted costunolide action on estrogen receptor-negative breast cancer cells--a comparison with normal breast cells.

Costunolide, a sesquiterpene lactone is a plant-derived secondary metabolite found to be present in most of the pharmacologically active herbs, being the cause for their medicinal values. The present study aims to evaluate the cytotoxic effect of costunolide isolated from Costus speciosus rhizome extract on MDA-MB-231 cells and explore its targeted action in comparison with its action on the normal breast cells (MCF 10A). The effect of costunolide on cell viability of the cells was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide viability assay. The targeted action of the compound was analyzed comparing the effectiveness of the compound to alter the protein expression levels of NF-κB subunits in the normal and the cancer cells using western blotting analysis. In silico studies were performed to predict the targeted interaction of costunolide with the NF-κB subunit proteins. Costunolide inhibited the cell viability of MDA-MB-231 cells in a dose-dependent manner leaving no significant change in the viability of the normal breast cells. The over expressed NF-κB subunits - p65, 52 and 100 in the cancer cells were found to be downregulated when treated with costunolide at an effective dose of 20 and 40 µM costunolide. In silico results provided stable interactions between costunolide and the target proteins, supporting the in vitro results in addition. Thus, costunolide derived from C. speciosus plant source elevates a fresh conviction for its use in breast cancer therapy for its cytotoxic efficacy and non-toxic nature.

Alteration of mitochondrial proteome due to activation of Notch1 signaling pathway.

The Notch signaling pathway, a known regulator of cell fate decisions, proliferation, and apoptosis, has recently been implicated in the regulation of glycolysis, which affects tumor progression. However, the impact of Notch on other metabolic pathways remains to be elucidated. To gain more insights into the Notch signaling and its role in regulation of metabolism, we studied the mitochondrial proteome in Notch1-activated K562 cells using a comparative proteomics approach. The proteomic study led to the identification of 10 unique proteins that were altered due to Notch1 activation. Eight of these proteins belonged to mitochondria-localized metabolic pathways like oxidative phosphorylation, glutamine metabolism, Krebs cycle, and fatty acid oxidation. Validation of some of these findings showed that constitutive activation of Notch1 deregulated glutamine metabolism and Complex 1 of the respiratory chain. Furthermore, the deregulation of glutamine metabolism involved the canonical Notch signaling and its downstream effectors. The study also reports the effect of Notch signaling on mitochondrial function and status of high energy intermediates ATP, NADH, and NADPH. Thus our study shows the effect of Notch signaling on mitochondrial proteome, which in turn affects the functioning of key metabolic pathways, thereby connecting an important signaling pathway to the regulation of cellular metabolism.