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COVID-19 caused by SARS-CoV-2 corona virus has become a global pandemic. In the absence of drugs and vaccine, and premises of time, efforts and cost required for their development, natural resources such as herbs are anticipated to provide some help and may also offer a promising resource for drug development. Here, we have investigated the therapeutic prospective of Ashwagandha for the COVID-19 pandemic. Nine withanolides were tested in silico for their potential to target and inhibit (i) cell surface receptor protein (TMPRSS2) that is required for entry of virus to host cells and (ii) viral protein (the main protease Mpro) that is essential for virus replication. We report that the withanolides possess capacity to inhibit the activity of TMPRSS2 and Mpro. Furthermore, withanolide-treated cells showed downregulation of TMPRSS2 expression and inhibition of SARS-CoV-2 replication in vitro, suggesting that Ashwagandha may provide a useful resource for COVID-19 treatment.

Activation of a telomere length maintenance mechanism (TMM), including telomerase and alternative lengthening of telomeres (ALT), is essential for replicative immortality of tumor cells, although its regulatory mechanisms are incompletely understood. We conducted a microRNA (miRNA) microarray analysis on isogenic telomerase positive (TEP) and ALT cancer cell lines. Amongst nine miRNAs that showed difference in their expression in TEP and ALT cancer cells in array analysis, miR-708 was selected for further analysis since it was consistently highly expressed in a large panel of ALT cells. miR-708 in TEP and ALT cancer cells was not correlated with C-circle levels, an established feature of ALT cells. Its overexpression induced suppression of cell migration, invasion, and angiogenesis in both TEP and ALT cells, although cell proliferation was inhibited only in TEP cells suggesting that ALT cells may have acquired the ability to escape inhibition of cell proliferation by sustained miR-708 overexpression. Further, cell proliferation regulation in TEP cells by miR708 appears to be through the CARF-p53 pathway. We demonstrate here that miR-708 (i) is the first miRNA shown to be differentially regulated in TEP and ALT cancer cells, (ii) possesses tumor suppressor function, and (iii) deregulates CARF and p21WAF1-mediated signaling to limit proliferation in TEP cells.

Serine/threonine-protein kinase B-raf (BRAF) plays a significant role in regulating cell division and proliferation through MAPK/ERK pathway. The constitutive expression of wild-type BRAF (BRAFWT) and its mutant forms, especially V600E (BRAFV600E), has been linked to multiple cancers. Various synthetic drugs have been approved and are in clinical trials, but most of them are reported to become ineffective within a short duration. Therefore, combinational therapy involving multiple drugs are often recruited for cancer treatment. However, they lead to toxicity and adverse side effects. In this computational study, we have investigated three natural compounds, namely Withaferin-A (Wi-A), Withanone (Wi-N) and Caffeic Acid Phenethyl ester (CAPE) for anti-BRAFWT and anti-BRAFV600E activity. We found that these compounds could bind stably at ATP-binding site in both BRAFWT and BRAFV600E proteins. In-depth analysis revealed that these compounds maintained the active conformation of wild-type BRAF protein by inducing αC-helix-In, DFG-In, extended activation segment and well-aligned R-spine residues similar to already known drugs Vemurafenib (VEM), BGB283 and Ponatinib. In terms of binding energy, among the natural compounds, CAPE showed better affinity towards both wild-type and V600E mutant proteins than the other two compounds. These data suggested that CAPE, Wi-A and Wi-N have potential to block constitutive autophosphorylation of BRAF and hence warrant in vitro and in vivo experimental validation., Graphical abstract Image 1, Highlights • Out of all the human cancers approximately 8% involve BRAF mutations. • The 40–50% of the commercialized drugs in the market are from the natural sources or inspired by it. • Three natural compounds Withaferin-A , Withanone and Caffeic acid phenethyl ester (CAPE) have been studied against BRAF. • CAPE binds with higher binding affinity with BRAF wild type protein and BRAF V600E mutant protein than other natural compounds.

Strategies for eradicating cancer stem cells (CSCs) are urgently required because CSCs are resistant to anticancer drugs and cause treatment failure, relapse and metastasis. Here, we show that photoactive functional nanocarbon complexes exhibit unique characteristics, such as homogeneous particle morphology, high water dispersibility, powerful photothermal conversion, rapid photoresponsivity and excellent photothermal stability. In addition, the present biologically permeable second near-infrared (NIR-II) light-induced nanocomplexes photo-thermally trigger calcium influx into target cells overexpressing the transient receptor potential vanilloid family type 2 (TRPV2). This combination of nanomaterial design and genetic engineering effectively eliminates cancer cells and suppresses stemness of cancer cells in vitro and in vivo. Finally, in molecular analyses of mechanisms, we show that inhibition of cancer stemness involves calcium-mediated dysregulation of the Wnt/β-catenin signalling pathway. The present technological concept may lead to innovative therapies to address the global issue of refractory cancers., Cancer stem cells (CSCs) are known to induce chemotherapy resistance, and cause tumour relapse and metastasis. Here, the authors develop photoactive nanocarbon complexes with second near-infrared photothermal ability to target cancer cells overexpressing the receptor TRPV2 and show it to suppress CSCs through dysregulation of the Wnt/β-catenin signalling pathway.

CARF (Collaborator of ARF) was discovered as an ARF-interacting protein that activated ARF-p53-p21(WAF1) signaling involved in cellular response to a variety of stresses, including oxidative, genotoxic, oncogenic, or telomere deprotection stresses, leading to senescence, growth arrest, or apoptosis. Of note, whereas suppression of CARF was lethal, its enrichment was associated with increased proliferation and malignant transformation of cells. These reports have predicted that CARF could serve as a multi-stress marker with a predictive value for cell fates. Here, we recruited various in vitro stress models and examined their effect on CARF expression using human normal fibroblasts. We demonstrate that CARF levels in stress and post-stress conditions could predict the fate of cells towards either death or enhanced proliferation and malignant transformation. We provide extensive molecular evidence that (i) CARF expression changes in response to stress, (ii) it modulates cell death or survival signaling and determines the fate of cells, and (iii) it may serve as a predictive measure of cellular response to stress and an important marker for biosafety. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s12192-020-01088-y) contains supplementary material, which is available to authorized users.

Coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus has become a global health emergency. Although new vaccines have been generated and being implicated, discovery and application of novel preventive and control measures are warranted. We aimed to identify compounds that may possess the potential to either block the entry of virus to host cells or attenuate its replication upon infection. Using host cell surface receptor expression (angiotensin-converting enzyme 2 (ACE2) and Transmembrane protease serine 2 (TMPRSS2)) analysis as an assay, we earlier screened several synthetic and natural compounds and identified candidates that showed ability to down-regulate their expression. Here, we report experimental and computational analyses of two small molecules, Mortaparib and MortaparibPlus that were initially identified as dual novel inhibitors of mortalin and PARP-1, for their activity against SARS-CoV-2. In silico analyses showed that MortaparibPlus, but not Mortaparib, stably binds into the catalytic pocket of TMPRSS2. In vitro analysis of control and treated cells revealed that MortaparibPlus caused down-regulation of ACE2 and TMPRSS2; Mortaparib did not show any effect. Furthermore, computational analysis on SARS-CoV-2 main protease (Mpro) that also predicted the inhibitory activity of MortaparibPlus. However, cell-based antiviral drug screening assay showed 30–60% viral inhibition in cells treated with non-toxic doses of either MortaparibPlus or Mortaparib. The data suggest that these two closely related compounds possess multimodal anti-COVID-19 activities. Whereas MortaparibPlus works through direct interactions/effects on the host cell surface receptors (ACE2 and TMPRSS2) and the virus protein (Mpro), Mortaparib involves independent mechanisms, elucidation of which warrants further studies.

Withania somnifera (Ashwagandha) is used in Indian traditional medicine, Ayurveda, and is believed to have a variety of health-promoting effects. The molecular mechanisms and pathways underlying these effects have not yet been sufficiently explored. In this study, we investigated the effect of Ashwagandha extracts and their major withanolides (withaferin A and withanone) on muscle cell differentiation using C2C12 myoblasts. We found that withaferin A and withanone and Ashwagandha extracts possessing different ratios of these active ingredients have different effects on the differentiation of C2C12. Withanone and withanone-rich extracts caused stronger differentiation of myoblasts to myotubes, deaggregation of heat- and metal-stress-induced aggregated proteins, and activation of hypoxia and autophagy pathways. Of note, the Parkinson’s disease model of Drosophila that possess a neuromuscular disorder showed improvement in their flight and climbing activity, suggesting the potential of Ashwagandha withanolides for the management of muscle repair and activity.

We previously performed a drug screening to identify a potential inhibitor of mortalin–p53 interaction. In four rounds of screenings based on the shift in mortalin immunostaining pattern from perinuclear to pan-cytoplasmic and nuclear enrichment of p53, we had identified MortaparibPlus (4-[(1E)-2-(2-phenylindol-3-yl)-1-azavinyl]-1,2,4-triazole) as a novel synthetic small molecule. In order to validate its activity and mechanism of action, we recruited Luminal-A breast cancer cells, MCF-7 (p53wild type) and T47D (p53L194F) and performed extensive biochemical and immunocytochemical analyses. Molecular analyses revealed that MortaparibPlus is capable of abrogating mortalin–p53 interaction in both MCF-7 and T47D cells. Intriguingly, upregulation of transcriptional activation function of p53 (as marked by upregulation of the p53 effector gene—p21WAF1—responsible for cell cycle arrest and apoptosis) was recorded only in MortaparibPlus-treated MCF-7 cells. On the other hand, MortaparibPlus-treated T47D cells exhibited hyperactivation of PARP1 (accumulation of PAR polymer and decrease in ATP levels) as a possible non-p53 tumor suppression program. However, these cells did not show full signs of either apoptosis or PAR-Thanatos. Molecular analyses attributed such a response to the inability of MortaparibPlus to disrupt the AIF–mortalin complexes, hence, AIF did not translocate to the nucleus to induce chromatinolysis and DNA degradation. These data suggested that the cancer cells possessing enriched levels of such complexes may not respond to MortaparibPlus. Taken together, we report the multimodal anticancer potential of MortaparibPlus that warrants further attention in laboratory and clinical studies.

Withaferin-A is a withanolide, predominantly present in Ashwagandha (Withania somnifera). It has been shown to possess anticancer activity in a variety of human cancer cells in vitro and in vivo. Molecular mechanism of such cytotoxicity has not yet been completely understood. Withaferin-A and Withanone were earlier shown to activate p53 tumor suppressor and oxidative stress pathways in cancer cells. 2,3-dihydro-3β-methoxy analogue of Withaferin-A (3βmWi-A) was shown to lack cytotoxicity and well tolerated at higher concentrations. It, on the other hand, protected normal cells against oxidative, chemical and UV stresses through induction of anti-stress and pro-survival signaling. We, in the present study, investigated the effect of Wi-A and 3βmWi-A on cell migration and metastasis signaling. Whereas Wi-A binds to vimentin and heterogeneous nuclear ribonucleoprotein K (hnRNP-K) with high efficacy and downregulates its effector proteins, MMPs and VEGF, involved in cancer cell metastasis, 3βmWi-A was ineffective. Consistently, Wi-A, and not 3βmWi-A, caused reduction in cytoskeleton proteins (Vimentin, N-Cadherin) and active protease (u-PA) that are essential for three key steps of cancer cell metastasis (EMT, increase in cell migration and invasion).

Cancer, an uncontrolled proliferation syndrome, is treated with synthetic chemotherapeutic drugs that are associated with severe adverse effects. Development and application of new natural compounds is warranted to deal with the exponentially increasing incidence of cancer worldwide. Keeping selective toxicity to cancer cells as a priority criterion, we developed a combination of Cucurbitacin B and Withanone, and analyzed its anticancer potential using non-small cell lung cancer cells. We demonstrate that the selective cytotoxicity of the combination, called CucWi-N, to cancer cells is mediated by induction of cellular senescence that was characterized by decrease in Lamin A/C, CDK2, CDK4, Cyclin D, Cyclin E, phosphorylated RB, mortalin and increase in p53 and CARF proteins. It compromised cancer cell migration that was mediated by decrease in mortalin, hnRNP-K, vascular endothelial growth factor, matrix metalloproteinase 2, and fibronectin. We provide in silico, molecular dynamics and experimental data to support that CucWi-N (i) possesses high capability to target mortalin–p53 interaction and hnRNP-K proteins, (ii) triggers replicative senescence and inhibits metastatic potential of the cancer cells, and (iii) inhibits tumor progression and metastasis in vivo. We propose that CucWi-N is a potential natural anticancer drug that warrants further mechanistic and clinical studies.

Cordyceps militaris possesses several compounds with medicinal properties, and is commonly used in traditional Chinese functional food and medicine for a variety of health benefits. Because of its rare occurrence in nature, the market demand for artificial C. militaris is on the rise. Furthermore, efforts to increase its bioactive ingredients have also been considered in research. In this study, we aimed to investigate the effect of fluoride on the growth and enrichment of bioactive compounds in C. militaris. A wide range of potassium fluoride concentrations (0, 0.001, 0.01, 0.1, and 1 mM) were added to the culture media as a source of fluoride during the cultivation of C. militaris fruiting bodies. The contents of fluorine and bioactive substances of the fruiting bodies in normal (NM) and fluorine-supplemented (FM) media were measured and compared. C. militaris raised in the growth medium supplemented with 0.01 mM potassium fluoride led to a 44.86% (1.55 ± 0.14 g/bottle) increase in biomass and a 23.43% (3161.38 ± 35.71 µg/g) increase in total carotenoid content in the fruiting bodies. Furthermore, a remarkable increase in superoxide dismutase-like activity (84.75 U/mg) and 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity (IC50 = 2.59 mg/mL) was recorded. In human cancer cell-based assays, C. militaris raised in FM caused stronger cytotoxicity, apoptosis, and cell cycle arrest in human osteosarcoma cells. These results demonstrated that a low dose of fluoride could stimulate the growth of C. militaris fruiting bodies and enhance the production of bioactive ingredients that possess useful antioxidant and anticancer activities.

SARS-CoV-2 outbreak in China in December 2019 and its spread as worldwide pandemic has been a major global health crisis. Extremely high infection and mortality rate has severely affected all sectors of life and derailed the global economy. While drug and vaccine development have been prioritized and have made significant progression, use of phytochemicals and herbal constituents is deemed as a low-cost, safer and readily available alternative. We investigated therapeutic efficacy of eight withanolides (derived from Ashwagandha) against the angiotensin-converting enzyme 2 (ACE2) proteins, a target cell surface receptor for SARS-CoV-2 and report results on the (i) computational analyses including binding affinity and stable interactions with ACE2, occupancy of ACE2 residues in making polar and nonpolar interactions with different withanolides/ligands and (2) in vitro mRNA and protein analyses using human cancer (A549, MCF7 and HSC3) cells. We found that among all withanolides, Withaferin-A, Withanone, Withanoside-IV and Withanoside-V significantly inhibited the ACE2 expression. Analysis of withanolides-rich aqueous extracts derived from Ashwagandha leaves and stem showed a higher ACE2 inhibitory potency of stem-derived extracts. Taken together, we demonstrated the inhibitory potency of Ashwagandha withanolides and its aqueous extracts against ACE2. Communicated by Ramaswamy H. Sarma

Poor bioavailability due to the inability to cross the cell membrane is one of the major reasons for the failure of a drug in clinical trials. We have used molecular dynamics simulations to predict the membrane permeability of natural drugs—withanolides (withaferin-A and withanone) that have similar structures but remarkably differ in their cytotoxicity. We found that whereas withaferin-A, could proficiently transverse through the model membrane, withanone showed weak permeability. The free energy profiles for the interaction of withanolides with the model bilayer membrane revealed that whereas the polar head group of the membrane caused high resistance for the passage of withanone, the interior of the membrane behaves similarly for both withanolides. The solvation analysis further revealed that the high solvation of terminal O5 oxygen of withaferin-A was the major driving force for its high permeability; it interacted with the phosphate group of the membrane that led to its smooth passage across the bilayer. The computational predictions were tested by raising and recruiting unique antibodies that react to withaferin-A and withanone. The time-lapsed analyses of control and treated cells demonstrated higher permeation of withaferin-A as compared to withanone. The concurrence between the computation and experimental results thus re-emphasised the use of computational methods for predicting permeability and hence bioavailability of natural drug compounds in the drug development process.

The anticancer activities of Withaferin-A (Wi-A) and Withanone (Wi-N) from Ashwagandha and Caffeic Acid Phenethyl Ester (CAPE) from honeybee propolis have been well documented. Here, we examined the binding potential of these natural compounds to inhibit the constitutive phosphorylation of epidermal growth factor receptors (EGFRs). Exon 20 insertion mutants of EGFR, which show resistance to various FDA approved drugs and are linked to poor prognosis of lung cancer patients, were the primary focus of this study. Apart from exon 20 insertion mutants, the potential of natural compounds to serve as ATP competitive inhibitors of wildtype protein and other common mutants of EGFR, namely L858R and exon19del, were also examined. The potential of natural compounds was compared to the positive controls such as erlotinib, TAS6417 and poziotinib. Similar to known inhibitors, Wi-A and Wi-N could displace and binds at the ATP orthosteric site of exon19del, L858R and exon20, while CAPE was limited to wildtype EGFR and exon 20 insertion mutants only. Moreover, the binding free energy of the natural drugs against EGFRs was also comparable to the positive controls. This computational study suggests that Wi-A and Wi-N have potential against multiple mutated EGFRs, warranting further in vitro and in vivo experiments.

Caffeic acid phenethyl ester (CAPE) is a key bioactive ingredient of honeybee propolis and is claimed to have anticancer activity. Since mortalin, a hsp70 chaperone, is enriched in a cancerous cell surface, we recruited a unique cell internalizing anti-mortalin antibody (MotAb) to generate mortalin-targeting CAPE nanoparticles (CAPE-MotAb). Biophysical and biomolecular analyses revealed enhanced anticancer activity of CAPE-MotAb both in in vitro and in vivo assays. We demonstrate that CAPE-MotAb cause a stronger dose-dependent growth arrest/apoptosis of cancer cells through the downregulation of Cyclin D1-CDK4, phospho-Rb, PARP-1, and anti-apoptotic protein Bcl2. Concomitantly, a significant increase in the expression of p53, p21WAF1, and caspase cleavage was obtained only in CAPE-MotAb treated cells. We also demonstrate that CAPE-MotAb caused a remarkably enhanced downregulation of proteins critically involved in cell migration. In vivo tumor growth assays for subcutaneous xenografts in nude mice also revealed a significantly enhanced suppression of tumor growth in the treated group suggesting that these novel CAPE-MotAb nanoparticles may serve as a potent anticancer nanomedicine.

Coronavirus disease 2019 (COVID-19) initiated in December 2019 in Wuhan, China and became pandemic causing high fatality and disrupted normal life calling world almost to a halt. Causative agent is a novel coronavirus called Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2/2019-nCoV). While new line of drug/vaccine development has been initiated world-wide, in the current scenario of high infected numbers, severity of the disease and high morbidity, repurposing of the existing drugs is heavily explored. Here, we used a homology-based structural model of transmembrane protease serine 2 (TMPRSS2), a cell surface receptor, required for entry of virus to the target host cell. Using the strengths of molecular docking and molecular dynamics simulations, we examined the binding potential of Withaferin-A (Wi-A), Withanone (Wi-N) and caffeic acid phenethyl ester to TPMRSS2 in comparison to its known inhibitor, Camostat mesylate. We found that both Wi-A and Wi-N could bind and stably interact at the catalytic site of TMPRSS2. Wi-N showed stronger interactions with TMPRSS2 catalytic residues than Wi-A and was also able to induce changes in its allosteric site. Furthermore, we investigated the effect of Wi-N on TMPRSS2 expression in MCF7 cells and found remarkable downregulation of TMPRSS2 mRNA in treated cells predicting dual action of Wi-N to block SARS-CoV-2 entry into the host cells. Since the natural compounds are easily available/affordable, they may even offer a timely therapeutic/preventive value for the management of SARS-CoV-2 pandemic. We also report that Wi-A/Wi-N content varies in different parts of Ashwagandha and warrants careful attention for their use. Communicated by Ramaswamy H. Sarma

The recent novel coronavirus, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2/2019-nCoV) has caused a large number of deaths around the globe. There is an urgent need to understand this...

Cell-based screening of bioactive compounds has served as an important gateway in drug discovery. In the present report, using human neuroblastoma cells and enrolling an extensive three-step screening of 57 phytochemicals, we have identified caffeic acid phenethyl ester (CAPE) as a potent neurodifferentiating natural compound. Analyses of control and CAPE-induced neurodifferentiated cells revealed: (i) modulation of several key proteins (NF200, MAP-2, NeuN, PSD95, Tuj1, GAP43, and GFAP) involved in neurodifferentiation process; and (ii) attenuation of neuronal stemness (HOXD13, WNT3, and Msh-2) and proliferation-promoting (CDC-20, CDK-7, and BubR1) proteins. We anticipated that the neurodifferentiation potential of CAPE may be beneficial for the treatment of neurodegenerative diseases and tested it using the Drosophila model of Alzheimer's disease (AD) and mice model of amnesia/loss of memory. In both models, CAPE exhibited improved disease symptoms and activation of physiological functions. Remarkably, CAPE-treated mice showed increased levels of neurotrophin-BDNF, neural progenitor marker-Nestin, and differentiation marker-NeuN, both in the cerebral cortex and hippocampus. Taken together, we demonstrate the differentiation-inducing and therapeutic potential of CAPE for neurodegenerative diseases.

We screened some phytochemicals for cytotoxic activity to human cancer cells and identified Soyasapogenol-A (Snol-A) as a potent candidate anti-cancer compound. Interestingly, Soyasapogenin-I (Snin-I) was ineffective. Viability assays endorsed toxicity of Snol-A to a wide variety of cancer cells. Of note, wild type p53 deficient cancer cells (SKOV-3 and Saos-2) also showed potent growth inhibitory effect. Molecular analyses demonstrated that it targets CARF yielding transcriptional upregulation of p21WAF1 (an inhibitor of cyclin-dependent kinases) and downregulation of its effector proteins, CDK2, CDK-4, Cyclin A and Cyclin D1. Targeting of CARF by Snol-A also caused (i) downregulation of pATR-Chk1 signaling leading to caspase-mediated apoptosis and (ii) inactivation of β-catenin/Vimentin/hnRNPK-mediated EMT signaling resulting in decrease in migration and invasion of cancer cells. In in vivo assays, Snol-A caused suppression of tumor growth in subcutaneous xenograft model and inhibited lung metastasis in tail vein injection model. Taken together, we demonstrate that Snol-A is a natural inhibitor of CARF and may be recruited as a potent anti-tumor and anti-metastasis compound for treatment of p53-deficient aggressive malignancies.

Environmental stress, exhaustive industrialization and the use of chemicals in our daily lives contribute to increasing incidence of cancer and other pathologies. Although the cancer treatment has revolutionized in last 2&ndash, 3 decades, shortcomings such as (i) extremely high cost of treatment, (ii) poor availability of drugs, (iii) severe side effects and (iv) emergence of drug resistance have prioritized the need of developing alternate natural, economic and welfare (NEW) therapeutics reagents. Identification and characterization of such anti-stress NEW drugs that not only limit the growth of cancer cells but also reprogram them to perform their specific functions are highly desired. We recruited rat glioma- and human neuroblastoma-based assays to explore such activities of resveratrol, a naturally occurring stilbenoid. We demonstrate that nontoxic doses of resveratrol protect cells against a variety of stresses that are largely involved in age-related brain pathologies. These included oxidative, DNA damage, metal toxicity, heat, hypoxia, and protein aggregation stresses. Furthermore, it caused differentiation of cells to functional astrocytes and neurons as characterized by the upregulation of their specific protein markers. These findings endorse multiple bioactivities of resveratrol and encourage them to be tested for their benefits in animal models and humans.

Background Mortalin is enriched in a large variety of cancers and has been shown to contribute to proliferation and migration of cancer cells in multiple ways. It has been shown to bind to p53 protein in cell cytoplasm and nucleus causing inactivation of its tumor suppressor activity in cancer cells. Several other activities of mortalin including mitochondrial biogenesis, ATP production, chaperoning, anti-apoptosis contribute to pro-proliferative and migration characteristics of cancer cells. Mortalin-compromised cancer cells have been shown to undergo apoptosis in in vitro and in vivo implying that it could be a potential target for cancer therapy. Methods We implemented a screening of a chemical library for compounds with potential to abrogate cancer cell specific mortalin-p53 interactions, and identified a new compound (named it as Mortaparib) that caused nuclear enrichment of p53 and shift in mortalin from perinuclear (typical of cancer cells) to pancytoplasmic (typical of normal cells). Biochemical and molecular assays were used to demonstrate the effect of Mortaparib on mortalin, p53 and PARP1 activities. Results Molecular homology search revealed that Mortaparib is a novel compound that showed strong cytotoxicity to ovarian, cervical and breast cancer cells. Bioinformatics analysis revealed that although Mortaparib could interact with mortalin, its binding with p53 interaction site was not stable. Instead, it caused transcriptional repression of mortalin leading to activation of p53 and growth arrest/apoptosis of cancer cells. By extensive computational and experimental analyses, we demonstrate that Mortaparib is a dual inhibitor of mortalin and PARP1. It targets mortalin, PARP1 and mortalin-PARP1 interactions leading to inactivation of PARP1 that triggers growth arrest/apoptosis signaling. Consistent with the role of mortalin and PARP1 in cancer cell migration, metastasis and angiogenesis, Mortaparib-treated cells showed inhibition of these phenotypes. In vivo tumor suppression assays showed that Mortaparib is a potent tumor suppressor small molecule and awaits clinical trials. Conclusion These findings report (i) the discovery of Mortaparib as a first dual inhibitor of mortalin and PARP1 (both frequently enriched in cancers), (ii) its molecular mechanism of action, and (iii) in vitro and in vivo tumor suppressor activity that emphasize its potential as an anticancer drug.

Honeybee propolis and its bioactive component, caffeic acid phenethyl ester (CAPE), are known for a variety of therapeutic potentials. By recruiting a cell-based reporter assay for screening of hypoxia-modulating natural drugs, we identified CAPE as a pro-hypoxia factor. In silico studies were used to probe the capacity of CAPE to interact with potential hypoxia-responsive proteins. CAPE could not dock into hypoxia inducing factor (HIF-1), the master regulator of hypoxia response pathway. On the other hand, it was predicted to bind to factor inhibiting HIF (FIH-1). The active site residue (Asp201) of FIH-1α was involved in hydrogen bond formation with CAPE and its analogue, caffeic acid methyl ester (CAME), especially in the presence of Fe and 2-oxoglutaric acid (OGA). We provide experimental evidence that the low doses of CAPE, that did not cause cytotoxicity or anti-migratory effect, activated HIF-1α and inhibited stress-induced protein aggregation, a common cause of age-related pathologies. Furthermore, by structural homology search, we explored and found candidate compounds that possess stronger FIH-1 binding capacity. These compounds could be promising candidates for modulating therapeutic potential of CAPE, and its recruitment in treatment of protein aggregation-based disorders. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s12192-018-0915-0) contains supplementary material, which is available to authorized users.

CARF (Collaborator of ARF)/CDKN2AIP was discovered as a novel ARF-binding protein. It has been established as an essential cell survival, p53-, and cell proliferation-regulatory protein. Although a moderate upregulation of CARF caused growth arrest and senescence, its excessively enriched levels were shown to facilitate aggressive proliferation and malignant transformation of cancer cells. Here, we examined the relevance of CARF levels in clinical tumors and found its amplification (both at gene and transcript levels) in a variety of invasive and metastatic malignancies. Consistent with the clinical readouts, enrichment of CARF in cancer cells promoted epithelial–mesenchymal transition (EMT). Cancer database and molecular analyses revealed that it activates Wnt/β-catenin signaling axis, as evident by enhanced nuclear localization and function of β-catenin marked by increased level of SNAIL1, SNAIL2, ZEB1, and TWIST1 and its downstream gene targets. Of note, targeted knockdown of CARF led to decrease in nuclear β-catenin and its key downstream effectors, involved in EMT progression. Consistent with this, CARF targeting in vivo either by naked siRNA or CARF shRNA harboring adeno-oncolytic virus caused suppression of tumor progression and lung metastasis. Taken together, we report clinical and therapeutic relevance of CARF in EMT and cancer invasiveness/metastasis, and propose it as a potent therapeutic target of aggressive cancers.

Withaferin-A (Wi-A) has been shown to possess anticancer activity. Molecular mechanism(s) of its action has not been fully resolved. We recruited low dose of Wi-A that caused slow growth arrest in cancer cells and was relatively safe for normal cells. Consistently, we detected nuclear translocation of nuclear factor kappa B (NFκB) and activation of p38MAPK selectively in cancer cells. Bioinformatics analyses revealed that Wi-A did not disrupt IKKα/IKKβ-Nemo complex that regulates NFκB activity. However, it caused moderate change in the conformation of IKKβ-Nemo interacting domain. Experimental data revealed increased level of phosphorylated IκBα in Wi-A-treated cells, suggesting an activation of IKK complex that was supported by nuclear translocation of NFκB. Molecular docking analysis showed that Wi-A did not disrupt; however, decreased the stability of the NFκB-DNA complex. It was supported by downregulation of DNA-binding and transcriptional activities of NFκB. Further analysis revealed that Wi-A caused upregulation of CARF (collaborator of ARF) demonstrating an activation of DNA damage oxidative stress response in both cancer and normal cells. In line with this, upregulation of p21WAF1, p16INK4A, and hypophosphorylated pRB and induction of senescence were observed demonstrating that Wi-A-induced senescence is mediated by multiple pathways in which CARF-mediated DNA damage and oxidative stress play a major role.

Brain and neuronal circuits constitute the most complex organ networks in human body. They not only control and coordinate functions of all other organs, but also represent one of the most-affected systems with stress, lifestyle and age. With global increase in aging populations, these neuropathologies have emerged as major concern for maintaining quality of life. Recent era has witnessed a surge in nutritional remediation of brain dysfunctions primarily by "nutraceuticals" that refer to functional foods and supplements with pharmacological potential. Specific dietary patterns with a balanced intake of carbohydrates, fatty acids, vitamins and micronutrients have also been ascertained to promote brain health. Dietary herbs and their phytochemicals with wide range of biological and pharmacological activities and minimal adverse effects have gained remarkable attention as neuro-nutraceuticals. Neuro-nutraceutical potentials of herbs are often expressed as effects on cognitive response, circadian rhythm, neuromodulatory, antioxidant and anti-inflammatory activities that are mediated by effects on gene expression, epigenetics, protein synthesis along with their turnover and metabolic pathways. Epidemiological and experimental evidence have implicated enormous applications of herbal supplementation in neurodegenerative and psychiatric disorders. The present review highlights the identification, experimental evidence and applications of some herbs including Bacopa monniera, Withania somnifera, Curcuma longa, Helicteres angustifolia, Undaria pinnatifida, Haematococcus pluvialis, and Vitis vinifera, as neuro-nutraceuticals.

Withania somniferais a highly valued medicinal plant in traditional home medicine and is known for a wide range of bioactivities. Its commercial cultivation is adversely affected by poor seed viability and germination. Infestation by various pests and pathogens, survival under unfavourable environmental conditions, narrow genetic base, and meager information regarding biosynthesis of secondary metabolites are some of the other existing challenges in the crop. Biotechnological interventions through organ, tissue, and cell culture provide promising options for addressing some of these issues.In vitropropagation facilitates conservation and sustainable utilization of the existing germplasms and broadening the genetic base. It would also provide means for efficient and rapid mass propagation of elite chemotypes and generating uniform plant material round the year for experimentation and industrial applications. The potential ofin vitrocell/organ cultures for the production of therapeutically valuable compounds and their large-scale production in bioreactors has received significant attention in recent years.In vitroculture system further provides distinct advantage for studying various cellular and molecular processes leading to secondary metabolite accumulation and their regulation. Engineering plants through genetic transformation and development of hairy root culture system are powerful strategies for modulation of secondary metabolites. The present review highlights the developments and sketches current scenario in this field.

Poor bioavailability due to the inability to cross the cell membrane is one of the major reasons for the failure of a drug in the clinical trials. We have used molecular dynamics simulations to predict the membrane permeability of natural drugs - withanolides (withaferin-A and withanone) that have similar structures but remarkably differ in their cytotoxicity. We found that withaferin-A, but not withanone, could proficiently transverse through the model membrane. The free energy profiles obtained were in accordance with the physico-chemical properties of the investigated drug molecules. It was observed that the polar head group of the bilayer exhibits high resistance for the passage of withanone as compared to withaferin-A, while the interior of the membrane behaves similarly for both withanolides. The solvation analysis revealed that the high solvation of terminal O5 oxygen of withaferin-A was the major driving force. The impact of the favorable interaction of terminal oxygen (O5) of withaferin-A with the phosphate of the membrane led to its smooth passage across the bilayer. The computational predictions were validated by raising and recruiting unique antibodies that react to withaferin-A and withanone. Further, the time-lapsed analyses of control and treated human normal and cancer cells, demonstrated proficient permeation of withaferin-A, but not withanone, through normal cells. These data strongly validated our computational method for predicting permeability and hence bioavailability of candidate compounds in the drug development process.Statement of significanceWhat determines the bioavailability of a drug? Does the ability to cross cell membrane determine this? A combined simulation/experimental study of the permeability of two natural drugs - withanolides (Wi-A and Wi-N) across the cell membrane was conducted. In the computational portion of the study, steered MD simulations were performed to investigate the propensity of the two molecules to permeate across the cell. It is found that Wi-A proceeds relatively simply across the cell compared to Wi-N. This trend was found to be consistent with experiment. This work is an important step towards understanding the molecular basis of permeability of natural drug molecules.

Mortalin is a widely studied stress chaperone that plays a significant role in diseases such as cancer, diabetes mellitus, liver cirrhosis, neurodegeneration and generalized aging. Based on these, the level of mortalin expression has been predicted to be an important and valuable diagnostic and prognostic marker. Conventional methods of protein analyses, such as Western blotting, immunohistochemistry or ELISA with antibodies provide specific, sensitive and useful outcomes. However, they are limited by lengthy and time-consuming protocols. Here, we present an upgrade to the existing ELISA techniques. We have prepared a conjugate of anti-mortalin antibody and luciferase enzyme that can be recruited for rapid (∼3 h) and quantitative detection of mortalin expression in a given biological sample.

Fucoxanthin is commonly found in marine organisms, however, to date, it has been one of the scarcely explored natural compounds. We investigated its activities in human cancer cell culture-based viability, migration, and molecular assays, and found that it possesses strong anticancer and anti-metastatic activities that work irrespective of the p53 status of cancer cells. In our experiments, fucoxanthin caused the transcriptional suppression of mortalin. Cell phenotype-driven molecular analyses on control and treated cells demonstrated that fucoxanthin caused a decrease in hallmark proteins associated with cell proliferation, survival, and the metastatic spread of cancer cells at doses that were relatively safe to the normal cells. The data suggested that the cancer therapy regimen may benefit from the recruitment of fucoxanthin, hence, it warrants further attention for basic mechanistic studies as well as drug development.

Stress, protein aggregation, and loss of functional properties of cells have been shown to contribute to several deleterious pathologies including cancer and neurodegeneration. The incidence of these pathologies has also been shown to increase with age and are often presented as evidence to the cumulative effect of stress and protein aggregation. Prevention or delay of onset of these diseases may prove to be unprecedentedly beneficial. In this study, we explored the anti-stress and differentiation-inducing potential of two marine bioactive carotenoids (astaxanthin and fucoxanthin) using rat glioma cells as a model. We found that the low (nontoxic) doses of both protected cells against UV-induced DNA damage, heavy metal, and heat-induced protein misfolding and aggregation of proteins. Their long-term treatment in glioma cells caused the induction of physiological differentiation into astrocytes. These phenotypes were supported by upregulation of proteins that regulate cell proliferation, DNA damage repair mechanism, and glial differentiation, suggesting their potential for prevention and treatment of stress, protein aggregation, and age-related pathologies.

Background Tumor suppressor p53 protein is frequently mutated in a large majority of cancers. These mutations induce local or global changes in protein structure thereby affecting its binding to DNA. The structural differences between the wild type and mutant p53 thus provide an opportunity to selectively target mutated p53 harboring cancer cells. Restoration of wild type p53 activity in mutants using small molecules that can revert the structural changes have been considered for cancer therapeutics. Methods We used bioinformatics and molecular docking tools to investigate the structural changes between the wild type and mutant p53 proteins (p53V143A, p53R249S, p53R273H and p53Y220C) and explored the therapeutic potential of Withaferin A and Withanone for restoration of wild type p53 function in cancer cells. Cancer cells harboring the specific mutant p53 proteins were used for molecular assays to determine the mutant or wild type p53 functions. Results We found that p53V143A mutation does not show any significant structural changes and was also refractory to the binding of withanolides. p53R249S mutation critically disturbed the H-bond network and destabilized the DNA binding site. However, withanolides did not show any selective binding to either this mutant or other similar variants. p53Y220C mutation created a cavity near the site of mutation with local loss of hydrophobicity and water network, leading to functionally inactive conformation. Mutated structure could accommodate withanolides suggesting their conformational selectivity to target p53Y220C mutant. Using human cell lines containing specific p53 mutant proteins, we demonstrated that Withaferin A, Withanone and the extract rich in these withanolides caused restoration of wild type p53 function in mutant p53Y220C cells. This was associated with induction of p21WAF-1-mediated growth arrest/apoptosis. Conclusion The study suggested that withanolides may serve as highly potent anticancer compounds for treatment of cancers harboring a p53Y220C mutation. Electronic supplementary material The online version of this article (10.1186/s13046-019-1099-x) contains supplementary material, which is available to authorized users.

Simple Summary Functional inactivation of tumour suppressor protein p53 is frequently found in a large variety of cancers. One of the mechanisms by which p53 is inactivated is through its interaction with mortalin protein that inhibits its translocation, and hence the function, in the nucleus. Abrogation of mortalin-p53 interaction has been suggested as a target for cancer therapy. We report here a novel multimodal small molecule, called MortaparibPlus, that causes growth arrest or apoptosis of cancer cells by abrogating mortalin-p53 interaction yielding reactivation of p53 function. It also causes upregulation of tumour suppressor protein p73, and inactivation of PARP1 and CARF proteins accounting for its multimodal anticancer activity. Abstract p53 has an essential role in suppressing the carcinogenesis process by inducing cell cycle arrest/apoptosis/senescence. Mortalin/GRP75 is a member of the Hsp70 protein family that binds to p53 causing its sequestration in the cell cytoplasm. Hence, p53 cannot translocate to the nucleus to execute its canonical tumour suppression function as a transcription factor. Abrogation of mortalin-p53 interaction and subsequent reactivation of p53’s tumour suppression function has been anticipated as a possible approach in developing a novel cancer therapeutic drug candidate. A chemical library was screened in a high-content screening system to identify potential mortalin-p53 interaction disruptors. By four rounds of visual assays for mortalin and p53, we identified a novel synthetic small-molecule triazole derivative (4-[(1E)-2-(2-phenylindol-3-yl)-1-azavinyl]-1,2,4-triazole, henceforth named MortaparibPlus). Its activities were validated using multiple bioinformatics and experimental approaches in colorectal cancer cells possessing either wild-type (HCT116) or mutant (DLD-1) p53. Bioinformatics and computational analyses predicted the ability of MortaparibPlus to competitively prevent the interaction of mortalin with p53 as it interacted with the p53 binding site of mortalin. Immunoprecipitation analyses demonstrated the abrogation of mortalin-p53 complex formation in MortaparibPlus-treated cells that showed growth arrest and apoptosis mediated by activation of p21WAF1, or BAX and PUMA signalling, respectively. Furthermore, we demonstrate that MortaparibPlus-induced cytotoxicity to cancer cells is mediated by multiple mechanisms that included the inhibition of PARP1, up-regulation of p73, and also the down-regulation of mortalin and CARF proteins that play critical roles in carcinogenesis. MortaparibPlus is a novel multimodal candidate anticancer drug that warrants further experimental and clinical attention.

Cancer is one of the leading causes of mortality worldwide that requires attention in terms of extensive study and research. Eradication of mortalin-p53 interaction that leads to the inhibition of transcriptional activation or blocking of p53 from functioning as a suppressor and induction of nuclear translocation of p53 can prove to be one of the useful approaches for cancer management.In this study, we used structure-based approach to target the p53-binding domain of mortalin in order to prevent mortalin-p53 complex formation. We screened compounds from ZINC database against the modeled mortalin protein using Glide virtual screening. The top two compounds, DTOM (ZINC 28639308) and TTOM (ZINC 38143676) with Glide score of -12.27 and -12.16, respectively, were identified with the potential to abrogate mortalin-p53 interaction. Finally, molecular dynamics simulations were used to analyze the dynamic stability of the ligand-bound complex and it was observed that residues Tyr196, Asn198, Val264 and Thr267 were involved in intermolecular interactions in both the simulated ligand-bound complexes, and thus, these residues may have a paramount role in stabilizing the binding of the ligands with the protein.These detailed insights can further facilitate the development of potent inhibitors against mortalin-p53 complex.

Rapidly increasing aging population and environmental stressors are the two main global concerns of the modern society. These have brought in light rapidly increasing incidence of a variety of pathological conditions including brain tumors, neurodegenerative & neuropsychiatric disorders, and new challenges for their treatment. The overlapping symptoms, complex etiology and lack of full understanding of the brain structure and function to-date further complicate these tasks. On the other hand, several herbal reagents with a long history of their use have been asserted to possess neurodifferentiation, neuroregenerative and neuroprotective potentials, and hence been recommended as supplement to enhance and maintain brain health and function. Although they have been claimed to function by holistic approach resulting in maintaining body homeostasis and brain health, there are not enough laboratory studies in support to these and mechanism(s) of such beneficial activities remain largely undefined. One such herb is Ashwagandha, also called "Queen of Ayurveda" for its popular use in Indian traditional home medicine because of its extensive benefits including anticancer, anti-stress and remedial potential for aging and neurodegenerative pathologies. However, active principles and underlying mechanism(s) of action remain largely unknown. Here we provide a review on the effects of Ashwagandha extracts and active principles, and underlying molecular mechanism(s) for brain pathologies. We highlight our findings on the nootropic potential of Ashwagandha leaves. The effects of Ashwagandha leaf extracts are multidimensional ranging from differentiation of neuroblastoma and glioma cells, reversal of Alzheimer and Parkinson's pathologies, protection against environmental neurotoxins and enhancement of memory.

In order to identify the cellular factors involved in human melanogenesis, we carried out shRNA-mediated loss-of-function screening in conjunction with induction of melanogenesis by 1-oleoyl-2-acetyl-glycerol (OAG) in human melanoma cells using biochemical and visual assays. Gene targets of the shRNAs (that caused loss of OAG-induced melanogenesis) and their pathways, as determined by bioinformatics, revealed involvement of proteins that regulate cell stress response, mitochondrial functions, proliferation, and apoptosis. We demonstrate, for the first time, that the mitochondrial stress chaperone mortalin is crucial for melanogenesis. Upregulation of mortalin was closely associated with melanogenesis in in vitro cell-based assays and clinical samples of keloids with hyperpigmentation. Furthermore, its knockdown resulted in compromised melanogenesis. The data proposed mortalin as an important protein that may be targeted to manipulate pigmentation for cosmetic and related disease therapeutics.

We had previously reported anticancer activity in the water extract (WEX) of Ashwagandha leaves, and identified Triethylene glycol (TEG) as an active tumor suppressor component. In this study, we investigated anti-migratory and anti-angiogenesis activities of WEX and TEG. We conducted in vitro and in vivo experiments using TEG, and its two derivatives, Triethyleneglycol dimethacrylate (TD-10), and Tetraethyleneglycol dimethacrylate (TD-11). The data revealed strong anticancer and anti-metastasis potentials in the derivatives. Non-toxic, anti-migratory doses of the derivatives showed inhibition of canonical Wnt/β-catenin axis and consequent downregulation of EMT-signaling proteins (Vimentin, MMPs and VEGF). These results endorse that the TD-10 and TD-11 have potential to safely put a check on the aggressiveness of the metastatic cells and therefore represent promising candidates for the treatment of metastatic cancers.

Skin color/pigmentation is regulated through melanogenesis process in specialized melanin-producing cells, melanocytes, involving multiple signaling pathways. It is highly influenced by intrinsic and extrinsic factors such as oxidative, ultraviolet radiations and other environmental stress conditions. Besides determining the color, it governs response and tolerance of skin to a variety of environmental stresses and pathological conditions including photodamage, hyperpigmentation, and skin cancer. Depigmenting reagents have been deemed useful not only for cosmetics but also for pigmentation-related pathologies. In the present study, we attempted modulation of 1-oleoyl-2-acetyl-glycerol- (OAG-) induced melanogenesis in human melanoma and primary melanocytes. In both cell types, OAG-induced melanogenesis was associated with increase in enhanced expression of melanin, tyrosinase, as well as stress chaperones (mortalin and HSP60) and Reactive Oxygen Species (ROS). Treatment with TXC (trans-4-(Aminomethyl) cyclohexanecarboxylic acid hexadecyl ester hydrochloride) and 5/40 natural compounds resulted in their reduction. The data proposed an important role of mortalin and oxidative stress in skin pigmentation and the use of TXC and natural extracts for modulation of pigmentation pathways in normal and pathological conditions.

Background Natural extracts and compounds used in traditional home medicine are known for their safety and a variety of health promoting and therapeutic potentials. In contrast to the single molecule mediated targets, the combinational therapies are preferred for their multi-functional and limited toxic regimens and may be useful for disease therapeutics as well as to increase the quality of life during a variety of environmental stresses. Purpose We aimed to combine the active ingredients of Chinese (Helicteres angustifolia) and Indian (Withania somnifera) ginsengs to develop a natural, efficient, and welfare combinatorial mixture with high anti-stress and glial differentiation potentials. Methods Using cultured cells as a model system, we developed a combination of active ingredients of Chinese (Cucurbitacin B [Cuc]) and Indian (Withanone [Wi-N]) ginsengs. Eleven chemical models of environmental stresses were used. Cytotoxicity studies were performed using human skin fibroblast for anti-stress and rat glioma cells for glial differentiation effects. Results We demonstrate that the novel combination of Cuc and Wi-N, CucWi-N, was non-toxic to normal cells. It caused stress protection in assays using normal human fibroblasts subjected to a variety of stresses. Of note, cells showed remarkable protection against oxidative and UV stresses and marked by decrease in DNA damage and reactive oxygen species. We examined and found the glial differentiation potential of CucWi-N in rat glioblastoma cells. CucWi-N clearly induced differentiation phenotype, well-marked with upregulation of GAP43, MAP2, and GFAP, which have been shown to play a key role in glial differentiation. Conclusion These data demonstrate anti-stress and glial differentiation potential of CucWi-N (a novel combination of Cuc and Wi-N) that could be recruited in nutraceutical and pharmaceutical avenues and hence warrant further evaluation and mechanistic studies.

Objective Short-term viability assays of cultured cells in 96-well plates are routinely used to determine the cytotoxicity or safety of drugs. These are often based on the formation of chromogen, generated selectively in viable cells. The innate problems of such short-term cell viability assays include (i) effect of drugs is determined by cell density (ii) some drugs have slow/gradual effect and hence may escape such assays, (iii) cell morphology that reveal significant hints to molecular signaling underlining the effect of drugs cannot be effectively captured, (iv) long-term effect on viability and clonogenic potential of cells cannot be determined and (v) herbal extracts often possess intrinsic color that interferes with spectrophotometer estimation. In light of the ease and importance of cell culture-based assessment of drug safety and cytotoxicity, we attempted to combine the conventional cell-based assays in a way that allows multiple readouts (quantitative and qualitative) from a single experiment, and avoids the drawbacks of color interference. Results We have established and validated (using 16 types of cultured mammalian cells) a Quantitative and Qualitative Cell Viability assay in 12-well cell culture plates. It overcomes several shortcomings as discussed above and allows long-term observations on cell morphology and clonogenicity. Electronic supplementary material The online version of this article (10.1186/s13104-018-3512-5) contains supplementary material, which is available to authorized users.

Stress is a state that triggers change in normal physiology and recognized by human body and brain as an unfavorable event causing concern, worry or anxiety. It may vary from physical, metabolic, physiological or emotional often culminating into wide range of ailments that may range from common cold, decline in functional efficacy of body systems or even cancer. Skin is the largest tissue of the body and makes the first interface with the environment. Skin color and characteristics are highly influenced by environment stress. A variety of natural compounds have been used for anti-stress and disease preventive potentials in worldwide traditional home medicine systems. They have recently attracted attention in research laboratories to dissect their mode of action to promote safe and economic drug development. We have earlier identified anti-stress and anti-aging activities in Withania somnifera, Helicteres angustifolia and honeybee propolis using human cultured normal and cancer cells. In the present study, we explored the effect of tamarind seed extracts prepared in water or 95% ethanol. In cell-based assays, we found that the extracts were safe to use in viable cells (in the range of 0.01-1.0%, for at least 4 weeks). Consistently, molecular studies revealed no effect on the expression/activity of cancer promoting proteins. We recruited oxidative stress models, such as, hydrogen peroxide (H2O2), ultraviolet radiation (UV) and diacylglycerol 1-oleoyl-2-acetyl-sn-glycerol (OAG). Investigation on anti-stress potential of the extracts revealed that they do not offer remarkable protection against stress caused by either H2O2 or UV, however, significantly compromised OAG-induced melanogenesis. The preliminary data warrant further investigations on the active components and mechanism of action to develop useful natural compounds/extracts for manipulation of melanogenesis that plays important role in response of cells to UV and its consequences including DNA damage, oxidative stress and related diseases.

2,3-Dihydro-3β-methoxy withaferin-A (3βmWi-A) is a natural withanolide that is structurally close to withaferin-A (Wi-A), is cytotoxic to human cancer cells, and is a candidate anticancer natural compound. Using cell-based biochemical, molecular, and imaging assays, we report that Wi-A and 3βmWi-A possess contrasting activities. Whereas Wi-A caused oxidative stress to normal cells, 3βmWi-A was well tolerated at even 10-fold higher concentrations. Furthermore, it promoted survival and protected normal cells against oxidative, UV radiation, and chemical stresses. We provide molecular evidence that 3βmWi-A induces antistress and pro-survival signaling through activation of the pAkt/MAPK pathway. We demonstrate that 3βmWi-A (i) contrary to Wi-A is safe and possesses stress-relieving activity, (ii) when given subsequent to a variety of stress factors including Wi-A, protects normal cells against their toxicity, and (iii) is a vital compound that may guard normal cells against the toxicity associated with various targeted therapeutic regimes in clinical practice.

Cancer is one of the most important healthcare matters, with the worst prognosis but the best possibilities for scientific development. It is likely to increase in the future and cause global havoc designating it as an epidemic. Cancer development requires urgent intervention. Past few decades have witnessed extensive research to challenge carcinogenesis. Treatment involving synthetic discipline is often associated with severe adverse effects, or even worsened prognosis. Accordingly, newer economic and patient friendly molecules are warranted. Many natural substances have proved their potential so far. Cucurbitacin B against cancer and other diseases has achieved towering popularity among the researchers around the world, as detailed in the below sections with summarized tables. In line with the fascinating role of cucurbitacin B against various types of cancers, through various molecular signaling pathways, it is justifiable to propose cucurbitacin B as a mainline chemotherapy before the onset and after the diagnosis of cancer.

CARF (Collaborator of ARF) was first identified as an ARF (Alternative Reading Frame, p14ARF)-interacting protein in a yeast two-hybrid interactive screening. Subsequently, it was shown to stabilize the p53-tumor suppressor protein in an ARF-dependent or -independent manner. It acts as a transcriptional repressor of HDM2 that exerts a negative feedback on p53 by its proteasomal-mediated degradation. CARF-driven control over p53-HDM2-p21WAF1 axis was shown to regulate cell proliferative fates. Cells with CARF-overexpression (CARF-OE) and superexpression (CARF-SE) showed growth arrest and pro-proliferative phenotypes, respectively. On the other hand, apoptosis was triggered in CARF-compromised cells. In the present review, we provide a comprehensive current understanding into the molecular mechanisms of CARF functions in regulation of DNA damage response, cell cycle checkpoints, cell survival and death signaling pathways. We discuss how thresh-hold of CARF level determines fate of cells to senescence and malignant transformation.

5'-Aza-2'-deoxycytidine (5-Aza-dC) is a demethylating drug that causes genome-wide hypomethylation resulting in the expression of several tumor suppressor genes causing growth arrest of cancer cells. Cancer is well established as a multifactorial disease and requires multi-module therapeutics. Search for new drugs and their approval by FDA takes a long time. Keeping this in view, research on new functions of FDA-approved anticancer drugs is desired to expand the list of multi-module functioning drugs for cancer therapy. In this study, we conducted an analysis for new functions of 5-Aza-dC by applying bio-chemo-informatics approach. The potential of 5-Aza-dC bioactivity was analyzed by PASS online and Molinspiration. Target proteins were predicted by SuperPred. The protein networks and biological processes were analyzed by Biological Networks using Gene Ontology tool, BINGO, based on BIOGRID database. Interactions between 5-Aza-dC and targeted proteins were examined by Autodoc Vina integrated into pyrx software. Induction of p53 by 5-Aza-dC was tested in vitro using cancer cells. Bioinformatics analyses predicted that 5-Aza-dC functions as a p53 inducer, radiosensitizer, and inhibitor of some enzymes. It was predicted to target proteins including MDM2, POLA1, POLB, and CXCR4 that are involved in the induction of DNA damage response and p53-HDM2-p21 signaling. In this study, we provide experimental evidence showing HDM2 is one of the targets of 5-AZA-dC leading to activation of p53 pathway and growth arrest of cells. Furthermore, we found that the combinatorial treatment of 5-AZA-dC with three other drugs caused drug resistance. We discuss that 5-Aza-dC-induced senescence is a multi-module drug that controls cell proliferation phenotype not only by proteins but also by noncoding miRNAs. Further studies are warranted to dissect these mechanisms and establish 5-Aza-dC as an effective multi-module anticancer reagent.

Mortalin/mtHsp70 is a member of Hsp70 family of proteins. Enriched in a large variety of cancers, it has been shown to contribute to the process of carcinogenesis by multiple ways including inactivation of tumor suppressor p53 protein, deregulation of apoptosis and activation of EMT signaling. In this study, we report that upregulation of mortalin contributes to cancer cell stemness. Several cancer cell stemness markers, such as ABCG2, OCT-4, CD133, ALDH1, CD9, MRP1 and connexin were upregulated in mortalin-overexpressing cells that showed higher ability to form spheroids. These cells also showed higher migration, and were less responsive to a variety of cancer chemotherapeutic drugs. Of note, knockdown of mortalin by specific shRNA sensitized these cells to all the drugs used in this study. We report that low doses of anti-mortalin molecules, MKT-077 and CAPE, also caused similar sensitization of cancer cells to chemotherapeutic drugs and hence are potential candidates for effective cancer chemotherapy.