Merger of Ayurveda and Tissue Culture-Based Functional Genomics: Inspirations from Systems Biology

Ayurveda is one of the ancient systems of health care of Indian origin. Roughly translated into "Knowledge of life", it is based on the use of natural herbs and herb products for therapeutic measures to boost physical, mental, social and spiritual harmony and improve quality of life. Although sheltered with long history and high trust, ayurveda principles have not entered laboratories and only a handful of studies have identified pure components and molecular pathways for its life-enhancing effects. In the post-genomic era, genome-wide functional screenings for targets for diseases is the most recent and practical approach. We illustrate here the merger of ayurveda and functional genomics in a systems biology scenario that reveals the pathway analysis of crude and active components and inspire ayurveda practice for health benefits, disease prevention and therapeutics

Folate receptor mediated targeting enhances selective cytotoxicity of Ashwagandha derived drugs to cancer cells

Background: Folate receptors (FRs) have been shown to be overexpressed on the surface of a variety of cancer cells and their expression are limited in normal cells and tissues. Since FR strongly binds to folic acid (FA), FA-functionalized nanocarriers have been proposed as a reliable strategy for delivery of anticancer drugs. We have earlier reported that the alcoholic extract of Ashwagandha leaves (i-Extract) and its major cytotoxic component, Withaferin A (Wi-A), have cancer cell killing activity. In the present study, we synthesized a FR-targeting i-Extract nanocomplex (FRi-ExNC) and a FR-targeting Wi-A nanocomposite (FRWi-ANC), by conjugating FA to polyethylene glycol and amphiphilic nanoframeworks, respectively. We investigated their anticancer potentials in in vitro and in vivo assays. Methods: Selective cellular uptake of FRi-ExNC and FRWi-ANC were evaluated by immunofluorescent microscopy. Cytotoxic effect of FRi-ExNC and FRWi-ANC in cancer cells were detected by assays including cell viability, apoptosis and biochemical determination of proteins involved in these phenotypes. The antitumor efficacy of FRi-ExNC and FRWi-ANC were investigated by in vivo tumor formation assays in nude mice. Results: We found that FRi-ExNC and FRWi-ANC caused stronger cytotoxicity as seen by induction of apoptosis. It was confirmed by cell cycle and protein expression analyses. In vivo tumor growth assays for subcutaneous xenografts in nude mice also revealed significantly enhanced suppression of tumor growth in the treated groups. Conclusions: Our results suggested that these two kinds of nanoparticles serve as useful nanomedical tools for selective targeting of drugs to the cancer cells and enhanced anticancer activity

Molecular insights to the dose-dependent activities of Ashwagandha extracts

Background: Stress is an inevitable component of life. Several herbs are known for their health supporting effects that range from treatment of stress, common cold to cancer. We investigated the dose-dependent effect of Ashwagandha (Withania somnifera) extracts on human normal and cancer cells, and have attempted to resolve the molecular mechanisms of their antistress activities. Methods: Ashwagandha extracts were chemically profiled by HPLC. Cytotoxicity was determined by viability assays. Biochemical and immunoimaging assays were performed using specific antibodies. Results: Human normal cells treated with low doses of the leaf extract or purified withanolides (Withaferin A or Withanone) showed no toxicity. Such non-toxic doses were selected for antistress, neurodifferentiation and neuroregenerative assays. We found that whereas normal cells exposed to oxidative and UV stresses showed poor viability/growth arrest/apoptosis, cells treated with low doses of Ashwagandha extracts were protected. Brain-derived cells exposed to glutamate and scopolamine stresses showed protection and strong differentiation as marked by expression of neurodifferentiation markers. Muscle-derived cells cultured in low doses of extract showed muscle differentiation as marked by expression of muscle differentiation markers. Most recently, using computational tools, we examined potential of Ashwagandha for anti-SARS-CoV2 virus activity, and found that most of the Ashwagandha Withanolides have potential to block cell surface receptors (ACE2 and TMPRSS2) that are involved in entry of virus to human cells. Furthermore, Ashwagandha treated cells showed decrease in ACE2 and TMPRSS2 expression suggesting its potential in blocking virus infection. Conclusion: Ashwagandha extracts and withanolides possess useful bioactivities

Cell-based experimental evidence to the anti-COVID-19 potential of Ashwagandha and honeybee propolis ingredients

Background: The COVID-19 pandemic emerged in December 2019 by a novel strain of SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2) has led to new endeavours in repurposing of existing drugs, anti-COVID-19 vaccine and drug development. Natural products, due to their general safety and wider availability, have attracted research and public attention. In this study, we report anti-COVID potential of compounds from honeybee propolis and Ayurvedic herb, Ashwagandha. Effect of active ingredients was studied on human cell surface receptors (ACE-2:Angiotensin Converting Enzyme-2/Spike protein and TMPRSS2:Transmembrane Protease Serine 2), critical for virus infection and virus main protease (Mpro, essential for virus replication), through molecular simulations and in vitro experiments. Methods: Structure-based computational analyses were performed to predict the effect of honeybee propolis (CAPE: Caffeic Acid Phenethyl Ester and ARC: Artepillin C), and Ashwagandha (Withanolides) ingredients on virus-host cell surface receptors. Cell-based assays were used to investigate the effect of these compounds on the expression level of the target proteins and virus replication. Results: Ashwagandha-derived nine withanolides were tested in silico for their potential to target and inhibit (i) ACE-2 and TMPRSS2 receptors (ii) viral main protease Mpro. We found that most withanolides possess capacity to bind to ACE-2, TMPRSS2 and Mpro . On the other hand, CAPE and ARC showed stable interactions at the active site of ACE2 and Mpro . ARC, but not CAPE, showed stable interaction with TMPRSS2. Human cells treated with withanolides, CAPE or ARC showed downregulation of both the receptors. Furthermore, celland PCR-based SARS-CoV-2 replication assays endorsed their antiviral activity. Conclusion: The findings suggest that the Ashwagandha-withanolides and honeybee propolis-derived compounds, CAPE, and ARC, could be helpful in the reduction of viral replication/infection, and hence warrant further experimental and clinical attention

Identification and characterization of anticancer potential of a novel small molecule, Mortaparibmild

Background: The development of new anticancer drugs and treatment modalities form a priority research field. The tumor suppressor protein p53 is frequently mutated or functionally inactivated in a large variety of cancers. Its inactivation by mortalin, a member of the heat shock 70 protein family, has been shown to contribute to carcinogenesis. The small molecule inhibitors of mortalin-p53 interactions have been shown to reactivate p53 yielding apoptosis/growth arrest in cancer cells. Therefore, abrogators of mortalin-p53 interaction have emerged as possible new therapeutic anticancer reagents. Methods: We performed chemical library screening based on the imaging of mortalin-p53 interaction, leading to the identification of a novel triazole derivative 4-[(4-amino-5-thiophen-2-yl-1,2,4-triazol-3-yl)sulfanylmethyl]-N-(4-methoxyphenyl)-1,3-thiazol-2-amine. Bioinformatics and experimental analyses were conducted to assess the anti-cancer potency of this molecule, named Mortaparibmild. Results: Mortaparibmild could bind to mortalin and p53 on their interaction sites. It caused downregulation of mortalin and PARP1 expression. However, a higher dose of Mortaparibmild was required for inducing apoptosis/growth arrest in cancer cells as compared to Mortaparib and MortaparibPlus, the previously reported molecules with similar properties [Elwakeel et. al. (2021) Cancers 13:3043; Sari et.al. (2021) Cancers 13:835 and Putri, et.al. (2019) J Exp Clin Cancer Res 38:1]. It was also effective for triggering apoptosis/growth arrest in p53null cancer cells suggesting its p53-independent activities. Molecular characterization of p53-dependent and independent Mortaparibmild activity and their relevance to cancer therapy will be discussed. Conclusion: Mortaparibmild is a new small molecule capable of inhibiting mortalin and PARP1 and inducing apoptosis in cancer cells

Protective Role of Ashwagandha Leaf Extract and Its Component Withanone on Scopolamine-Induced Changes in the Brain and Brain-Derived Cells

BACKGROUND:Scopolamine is a well-known cholinergic antagonist that causes amnesia in human and animal models. Scopolamine-induced amnesia in rodent models has been widely used to understand the molecular, biochemical, behavioral changes, and to delineate therapeutic targets of memory impairment. Although this has been linked to the decrease in central cholinergic neuronal activity following the blockade of muscarinic receptors, the underlying molecular and cellular mechanism(s) particularly the effect on neuroplasticity remains elusive. In the present study, we have investigated (i) the effects of scopolamine on the molecules involved in neuronal and glial plasticity both in vivo and in vitro and (ii) their recovery by alcoholic extract of Ashwagandha leaves (i-Extract). METHODOLOGY/PRINCIPAL FINDINGS:As a drug model, scopolamine hydrobromide was administered intraperitoneally to mice and its effect on the brain function was determined by molecular analyses. The results showed that the scopolamine caused downregulation of the expression of BDNF and GFAP in dose and time dependent manner, and these effects were markedly attenuated in response to i-Extract treatment. Similar to our observations in animal model system, we found that the scopolamine induced cytotoxicity in IMR32 neuronal and C6 glioma cells. It was associated with downregulation of neuronal cell markers NF-H, MAP2, PSD-95, GAP-43 and glial cell marker GFAP and with upregulation of DNA damage--γH2AX and oxidative stress--ROS markers. Furthermore, these molecules showed recovery when cells were treated with i-Extract or its purified component, withanone. CONCLUSION:Our study suggested that besides cholinergic blockade, scopolamine-induced memory loss may be associated with oxidative stress and Ashwagandha i-Extract, and withanone may serve as potential preventive and therapeutic agents for neurodegenerative disorders and hence warrant further molecular analyses

Stress-induced changes in CARF expression determine growth arrest, apoptosis, or malignant transformation in cultured human cells: Molecular evidence and its application

Background: CARF (Collaborator of ARF)/CDKN2AIP is an essential protein, first cloned as a binding partner of ARF. It was subsequently shown to interact with p53, HDM2 proteins and regulate growth arrest and apoptosis by its multimodal mechanism of action. Over-expression of CARF caused senescence like growth arrest of cells, its knock-down triggered apoptosis. Intriguingly, malignantly transformed cells showed high level of CARF expression. Based on these findings, we hypothesized that level of CARF expression may be a key determinant of cell proliferation fates; where an increase in its levels causes growth arrest/senescence, but beyond a threshold it activates carcinogenesis. Methods: We utilized in vitro cell culture models using retrovirus-driven expression of CARF to achieve over-expression and super-expression of CARF. Analysis of CARF levels was undertaken by biochemical and imaging protocols. Cells exposed to a variety of stresses including physiological, environmental, oxidative, radiation and chemotherapeutics was examined for CARF expression and corresponding cell proliferation fates. Results: Induction of Senescence was seen in cells over-expressing CARF. On the other hand, cells compromised for CARF showed apoptosis, and the ones with super-expression of CARF exhibited malignant transformation. CARF expression analysis in these experimental models endorsed the concept of cell-fate determining role of CARF. Conclusions: We present molecular evidence of the bridging role of CARF in stress-aging-cancer phenotypes and its application in pharmaceuticals and nutraceuticals as a diagnostic and prognostic marker for stress and cancer treatments

Embelin inhibits TNF-α converting enzyme and cancer cell metastasis: molecular dynamics and experimental evidence

BACKGROUND: Embelin, a quinone derivative, is found in the fruits of Embelia ribes Burm (Myrsinaceae). It has been shown to have a variety of therapeutic potentials including anthelmintic, anti-tumor, anti-diabetic, anti-bacterial and anti-inflammation. Inflammation is an immunological response to external harmful stimuli and is regulated by an endogenous pyrogen and pleiotropic pro-inflammatory cytokine, tumor necrosis factor alpha (TNF-α). TNF-α production has been implicated in a variety of other human pathologies including neurodegeneration and cancer. Several studies have shown that the anti-inflammatory activity of embelin is mediated by reduction in TNF-α. The latter is synthesized as a membrane anchored protein (pro-TNF-α); the soluble component of pro-TNF-α is then released into the extracellular space by the action of a protease called TNF-α converting enzyme (TACE). TACE, hence, has been proposed as a therapeutic target for inflammation and cancer. METHODS: We used molecular docking and experimental approaches to investigate the docking potential and molecular effects of embelin to TACE and human cancer cell characteristics, respectively. RESULTS: We demonstrate that embelin is a potential inhibitor of TACE. Furthermore, in vitro studies revealed that it inhibits malignant properties of cancer cells through inactivation of metastatic signaling molecules including MMPs, VEGF and hnRNP-K in breast cancer cells. CONCLUSION: Based on the molecular dynamics and experimental data, embelin is proposed as a natural anti-inflammatory and anticancer drug

MortaparibPlus- A Novel Anticancer Small Molecule Abrogating Mortalin-p53 Interaction in Cancer Cells

Background. The cessation of tumor cell growth through cell cycle arrest and apoptosis is determined by p53, a tumor suppressor protein. However, the interaction between mortalin-p53 within cytoplasm/nucleus leads to the inactivation of p53 transcriptional activation function. The disruption of mortalin-p53 complex has been suggested as an approach for developing a potential anticancer drug. Methods. A screening of a high-content chemical library was performed to determine a molecule with mortalin-p53-interaction disrupting characteristics. After four-rounds of visual assays, we discovered a triazole derivative (4-[(1E)-2-(2-phenylindol-3-yl)-1-azavinyl]-1,2,4-triazole, named MortaparibPlus) with a potential ability of disrupting mortalin-p53-complex. In this study, we recruited two types of cells (different p53 status and point mutation), Colorectal Cancer Cells [HCT116 (p53WT) and DLD-1 (p53 (p53S241F)] and Luminal A Breast Cancer [MCF-7 (p53WT) and T47D (p53L194F)]. We further validated the activity of MortaparibPlus by bioinformatics/experimental analyses. Results. Through bioinformatics analysis, we discovered that MortaparibPlus has potential to block the binding site of mortalin on p53, thus, preventing the formation of mortalin-p53 complex. Immunoprecipitation analyses showed that MortaparibPlus abrogated the mortalin-p53 complex formation and caused growth arrest/apoptosis (via activation of p21WAF1, BAX, and PUMA) in HCT116, DLD-1, and MCF-7 cells. Furthermore, MortaparibPlus posed a cytotoxic effect to cancer cells through various mechanisms (inhibition of PARP1, up-regulation of p73 proteins, downregulation of mortalin and CARF proteins). In contrast, we found that, despite the hyperactivation of PARP1 (PAR accumulation and loss of ATP) as an alternative tumor suppression mechanism, MortaparibPlus-treated T47D cells exhibited signs of neither complete apoptosis nor PAR-Thanatos. Such response was associated with the failure of MortaparibPlus to inhibit the formation of AIF-mortalin complexes. Conclusions. MortaparibPlus is proposed as a potential multimodal small molecule for cancer treatment that requires further extensive laboratory and clinical studies