Stress-induced changes in CARF expression serve as a quantitative predictive measure of cell proliferation fate.

Discovery of CARF (Collaborator of ARF)/CDKN2AIP as an ARF-interacting protein that promotes ARF-p53-p21WAF1 signaling and cellular senescence, initially established its role in genomic stress. Multiple reports further unraveled its role in regulation of senescence, growth arrest, apoptosis, or malignant transformation of cells in response to a variety of stress conditions in cultured human cells. It has been established as an essential protein. Whereas CARF-compromised cells undergo apoptosis, its enrichment has been recorded in a variety of cancer cells and has been associated with malignant transformation. We earlier demonstrated its role in stress-induced cell phenotypes that ranged from growth arrest, apoptosis, or malignant transformation. In the present study, we assessed the molecular mechanism of quantitative impact of change in CARF expression level on these cell fates. Stress-induced changes in CARF expression were assessed quantitatively with proteins involved in proteotoxicity, oxidative, genotoxic, and cytotoxic stress. These comparative quantitative analyses confirmed that (i) CARF responds to diverse stresses in a quantitative manner, (ii) its expression level serves as a reliable predictive measure of cell fates (iii) it correlates more with the DNA damage and MDA levels than the oxidative and proteotoxic signatures and (iv) CARF-expression based quantitative assay may be recruited for stress diagnostic applications.

Withanolide Derivative 2,3-Dihydro-3ß-methoxy Withaferin-A Modulates the Circadian Clock via Interaction with RAR-Related Orphan Receptor α (RORa).

Withanolide derivatives have anticancer, anti-inflammatory, and other functions and are components of Indian traditional Ayurvedic medicine. Here, we found that 2,3-dihydro-3ß-methoxy withaferin-A (3ßmWi-A), a derivative of withaferin-A (Wi-A) belonging to a class of withanolides that are abundant in Ashwagandha (Withania somnifera), lengthened the period of the circadian clock. This compound dose-dependently elongated circadian rhythms in Sarcoma 180 cancer cells and in normal fibroblasts including NIH3T3 and spontaneously immortalized mouse embryonic fibroblasts (MEF). Furthermore, 3ßmWi-A dose-dependently upregulated the mRNA expression and promoter activities of Bmal1 after dexamethasone stimulation and of the nuclear orphan receptors, Rora and Nr1d1, that comprise the stabilization loop for Bmal1 oscillatory expression. We showed that 3ßmWi-A functions as an inverse agonist for RORa with an IC50 of 11.3 µM and that 3ßmWi-A directly, but weakly, interacts with RORa (estimated dissociation constant [Kd], 5.9 µM). We propose that 3ßmWi-A is a novel modulator of circadian rhythms.

Molecular Insights into the Antistress Potentials of Brazilian Green Propolis Extract and Its Constituent Artepillin C.

Propolis, also known as bee-glue, is a resinous substance produced by honeybees from materials collected from plants they visit. It contains mixtures of wax and bee enzymes and is used by bees as a building material in their hives and by humans for different purposes in traditional healthcare practices. Although the composition of propolis has been shown to depend on its geographic location, climatic zone, and local flora; two largely studied types of propolis: (i) New Zealand and (ii) Brazilian green propolis have been shown to possess Caffeic Acid Phenethyl Ester (CAPE) and Artepillin C (ARC) as the main bioactive constituents, respectively. We have earlier reported that CAPE and ARC possess anticancer activities, mediated by abrogation of mortalin-p53 complex and reactivation of p53 tumor suppressor function. Like CAPE, Artepillin C (ARC) and the supercritical extract of green propolis (GPSE) showed potent anticancer activity. In this study, we recruited low doses of GPSE and ARC (that did not affect either cancer cell proliferation or migration) to investigate their antistress potential using in vitro cell based assays. We report that both GPSE and ARC have the capability to disaggregate metal- and heat-induced aggregated proteins. Metal-induced aggregation of GFP was reduced by fourfold in GPSE- as well as ARC-treated cells. Similarly, whereas heat-induced misfolding of luciferase protein showed 80% loss of activity, the cells treated with either GPSE or ARC showed 60-80% recovery. Furthermore, we demonstrate their pro-hypoxia (marked by the upregulation of HIF-1α) and neuro-differentiation (marked by differentiation morphology and upregulation of expression of GFAP, ß-tubulin III, and MAP2). Both GPSE and ARC also offered significant protection against oxidative stress and, hence, may be useful in the treatment of old age-related brain pathologies.

Novel role of mortalin in attenuating HIV-1 Tat-mediated astrogliosis.

BACKGROUND: In human immunodeficiency virus-1 (HIV-1) infection, activation of astrocytes induces imbalance in physiological functions due to perturbed astrocytic functions that unleashes toxicity on neurons. This leads to inflammatory response finally culminating into neurocognitive dysfunction. In neuroAIDS, HIV-1 protein, transactivator of transcription (Tat) is detected in the cerebrospinal fluid of infected patients. Mortalin, a multifunctional protein, has anti-inflammatory role following its activation in various stress conditions. Recent studies demonstrate downregulation of mortalin in neurodegenerative diseases. Here, we explored the mechanisms of mortalin in modulating HIV-1 Tat-mediated neuroinflammation. METHODS: Expression of mortalin in autopsy section in normal and diseased individuals were examined using immunohistochemistry. To decipher the role of mortalin in HIV-1 Tat-induced activation, human fetal brain-derived astrocytes were transiently transfected with Tat and mortalin using expression vectors. HIV-1 Tat-mediated damage was analyzed using RT-PCR and western blotting. Modulatory role of mortalin was examined by coexpressing it with Tat, followed by examination of mitochondrial morphodynamics using biochemical assay and confocal and electron microscopy. Extracellular ATP release was monitored using luciferase assay. Neuroinflammation in astrocytes was examined using flow cytometry, dye based study, immunocytochemistry, immunoprecipitation, and western blotting. Indirect neuronal damage was also analyzed. RESULTS: HIV-1 Tat downregulates the expression of mortalin in astrocytes, and this is corroborated with autopsy sections of HIV-1 patients. We found that overexpression of mortalin with Tat reduced inflammation and also rescued astrocytic-mediated neuronal death. Using bioinformatics, we discovered that binding of mortalin with Tat leads to Tat degradation and rescues the cell from neuroinflammation. Blocking of proteosomal pathway rescued the Tat degradation and revealed the ubiquitination of Tat. CONCLUSION: Overall, our data demonstrated the protective role of mortalin in combating HIV-1 Tat-mediated damage. We also showed that mortalin could degrade Tat through direct binding with HIV-1 Tat. Overexpression of mortalin in the presence of Tat could significantly reduce cytotoxic effects of Tat in astrocytes. Indirect neuronal death was also found to be rescued. Our in vitro findings were validated as we found attenuated expression of mortalin in the autopsy sections of HIV-1 patients.

Bioinformatics and Molecular Insights to Anti-Metastasis Activity of Triethylene Glycol Derivatives.

The anti-metastatic and anti-angiogenic activities of triethylene glycol derivatives have been reported. In this study, we investigated their molecular mechanism(s) using bioinformatics and experimental tools. By molecular dynamics analysis, we found that (i) triethylene glycol dimethacrylate (TD-10) and tetraethylene glycol dimethacrylate (TD-11) can act as inhibitors of the catalytic domain of matrix metalloproteinases (MMP-2, MMP-7 and MMP-9) by binding to the S1' pocket of MMP-2 and MMP-9 and the catalytic Zn ion binding site of MMP-7, and that (ii) TD-11 can cause local disruption of the secondary structure of vascular endothelial growth factor A (VEGFA) dimer and exhibit stable interaction at the binding interface of VEGFA receptor R1 complex. Cell-culture-based in vitro experiments showed anti-metastatic phenotypes as seen in migration and invasion assays in cancer cells by both TD-10 and TD-11. Underlying biochemical evidence revealed downregulation of VEGF and MMPs at the protein level; MMP-9 was also downregulated at the transcriptional level. By molecular analyses, we demonstrate that TD-10 and TD-11 target stress chaperone mortalin at the transcription and translational level, yielding decreased expression of vimentin, fibronectin and hnRNP-K, and increase in extracellular matrix (ECM) proteins (collagen IV and E-cadherin) endorsing reversal of epithelial-mesenchymal transition (EMT) signaling.

Rat Glioma Cell-Based Functional Characterization of Anti-Stress and Protein Deaggregation Activities in the Marine Carotenoids, Astaxanthin and Fucoxanthin.

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.

Marine Carotenoid Fucoxanthin Possesses Anti-Metastasis Activity: Molecular Evidence.

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.

Molecular dynamics-based identification of novel natural mortalin-p53 abrogators as anticancer agents.

INTRODUCTION: 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. RESULTS: 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. CONCLUSION: These detailed insights can further facilitate the development of potent inhibitors against mortalin-p53 complex.

2,3-Dihydro-3ß-methoxy Withaferin-A Protects Normal Cells against Stress: Molecular Evidence of Its Potent Cytoprotective Activity.

2,3-Dihydro-3ß-methoxy withaferin-A (3ßmWi-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 3ßmWi-A possess contrasting activities. Whereas Wi-A caused oxidative stress to normal cells, 3ßmWi-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 3ßmWi-A induces antistress and pro-survival signaling through activation of the pAkt/MAPK pathway. We demonstrate that 3ßmWi-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.

Functional significance of point mutations in stress chaperone mortalin and their relevance to Parkinson disease.

Mortalin/mtHsp70/Grp75 (mot-2), a heat shock protein 70 family member, is an essential chaperone, enriched in cancers, and has been shown to possess pro-proliferative and anti-apoptosis functions. An allelic form of mouse mortalin (mot-1) that differs by two amino acids, M618V and G624R, in the C terminus substrate-binding domain has been reported. Furthermore, genome sequencing of mortalin from Parkinson disease patients identified two missense mutants, R126W and P509S. In the present study, we investigated the significance of these mutations in survival, proliferation, and oxidative stress tolerance in human cells. Using mot-1 and mot-2 recombinant proteins and specific antibodies, we performed screening to find their binding proteins and then identified ribosomal protein L-7 (RPL-7) and elongation factor-1 α (EF-1α), which differentially bind to mot-1 and mot-2, respectively. We demonstrate that mot-1, R126W, or P509S mutant (i) lacks mot-2 functions involved in carcinogenesis, such as p53 inactivation and hTERT/hnRNP-K (heterogeneous nuclear ribonucleoprotein K) activation; (ii) causes increased level of endogenous oxidative stress; (iii) results in decreased tolerance of cells to exogenous oxidative stress; and (iv) shows differential binding and impact on the RPL-7 and EF-1α proteins. These factors may mediate the transformation of longevity/pro-proliferative function of mot-2 to the premature aging/anti-proliferative effect of mutants, and hence may have significance in cellular aging, Parkinson disease pathology, and prognosis.

Fate of bone marrow mesenchymal stromal cells following autologous transplantation in a rabbit model of osteonecrosis.

BACKGROUND AIMS: Internalizing quantum dots (i-QDs) are a useful tool for tracking cells in vivo in models of tissue regeneration. We previously synthesized i-QDs by conjugating QDs with a unique internalizing antibody against a heat shock protein 70 family stress chaperone. In the present study, i-QDs were used to label rabbit mesenchymal stromal cells (MSCs) that were then transplanted into rabbits to assess differentiation potential in an osteonecrosis model. METHODS: The i-QDs were taken up by bone marrow-derived MSCs collected from the iliac of 12-week-old Japanese white rabbits that were positive for cluster of differentiation (CD)81 and negative for CD34 and human leukocyte antigen DR. The average rate of i-QD internalization was 93.3%. At 4, 8, 12, and 24 weeks after transplantation, tissue repair was evaluated histologically and by epifluorescence and electron microscopy. RESULTS: The i-QDs were detected at the margins of the drill holes and in the necrotized bone trabecular. There was significant colocalization of the i-QD signal in transplanted cells and markers of osteoblast and mineralization at 4, 8, and 12 weeks post-transplantation, while i-QDs were detected in areas of mineralization at 12 and 24 weeks post-transplantation. Moreover, i-QDs were observed in osteoblasts in regenerated tissue by electron microscopy, demonstrating that the tissue was derived from transplanted cells. CONCLUSION: These results indicate that transplanted MSCs can differentiate into osteoblasts and induce tissue repair in an osteonecrosis model and can be tracked over the long term by i-QD labeling.

Collaborator of ARF (CARF) regulates proliferative fate of human cells by dose-dependent regulation of DNA damage signaling.

Collaborator of ARF (CARF) has been shown to directly bind to and regulate p53, a central protein that controls tumor suppression via cellular senescence and apoptosis. However, the cellular functions of CARF and the mechanisms governing its effect on senescence, apoptosis, or proliferation are still unknown. Our previous studies have shown that (i) CARF is up-regulated during replicative and stress-induced senescence, and its exogenous overexpression caused senescence-like growth arrest of cells, and (ii) suppression of CARF induces aneuploidy, DNA damage, and mitotic catastrophe, resulting in apoptosis via the ATR/CHK1 pathway. In the present study, we dissected the cellular role of CARF by investigating the molecular pathways triggered by its overexpression in vitro and in vivo. We found that the dosage of CARF is a critical factor in determining the proliferation potential of cancer cells. Most surprisingly, although a moderate level of CARF overexpression induced senescence, a very high level of CARF resulted in increased cell proliferation. We demonstrate that the level of CARF is crucial for DNA damage and checkpoint response of cells through ATM/CHK1/CHK2, p53, and ERK pathways that in turn determine the proliferative fate of cancer cells toward growth arrest or proproliferative and malignant phenotypes. To the best of our knowledge, this is the first report that demonstrates the capability of a fundamental protein, CARF, in controlling cell proliferation in two opposite directions and hence may play a key role in tumor biology and cancer therapeutics.

Identification and functional characterization of nuclear mortalin in human carcinogenesis.

The Hsp70 family protein mortalin is an essential chaperone that is frequently enriched in cancer cells and exists in various subcellular sites, including the mitochondrion, plasma membrane, endoplasmic reticulum, and cytosol. Although the molecular mechanisms underlying its multiple subcellular localizations are not yet clear, their functional significance has been revealed by several studies. In this study, we examined the nuclear fractions of human cells and found that the malignantly transformed cells have more mortalin than the normal cells. We then generated a mortalin mutant that lacked a mitochondrial targeting signal peptide. It was largely localized in the nucleus, and, hence, is called nuclear mortalin (mot-N). Functional characterization of mot-N revealed that it efficiently protects cancer cells against endogenous and exogenous oxidative stress. Furthermore, compared with the full-length mortalin overexpressing cancer cells, mot-N derivatives showed increased malignant properties, including higher proliferation rate, colony forming efficacy, motility, and tumor forming capacity both in in vitro and in vivo assays. We demonstrate that mot-N promotes carcinogenesis and cancer cell metastasis by inactivation of tumor suppressor protein p53 functions and by interaction and functional activation of telomerase and heterogeneous ribonucleoprotein K (hnRNP-K) proteins.

Molecular characterization of collaborator of ARF (CARF) as a DNA damage response and cell cycle checkpoint regulatory protein.

CARF is an ARF-binding protein that has been shown to regulate the p53-p21-HDM2 pathway. CARF overexpression was shown to cause growth arrest of human cancer cells and premature senescence of normal cells through activation of the p53 pathway. Because replicative senescence involves permanent withdrawal from the cell cycle in response to DNA damage response-mediated signaling, in the present study we investigated the relationship between CARF and the cell cycle and whether it is involved in the DNA damage response. We demonstrate that the half-life of CARF protein is less than 60 min, and that in cycling cells CARF levels are highest in G2 and early prophase. Serially passaged normal human skin and stromal fibroblasts showed upregulation of CARF during replicative senescence. Induction of G1 growth arrest and senescence by a variety of drugs was associated with increase in CARF expression at the transcriptional and translational level and was seen to correlate with increase in DNA damage response and checkpoint proteins, ATM, ATR, CHK1, CHK2, γH2AX, p53 and p21. Induction of growth arrest by oncogenic RAS and shRNA-mediated knockdown of TRF2 in cancer cells also caused upregulation of CARF. We conclude that CARF is associated with DNA damage response and checkpoint signaling pathways.

Biotechnological interventions in Withania somnifera (L.) Dunal.

Withania somnifera is one of the most valued plants and is extensively used in Indian, Unani, and African systems of traditional medicine. It possess a wide array of therapeutic properties including anti-arthritic, anti-aging, anti-cancer, anti-inflammatory, immunoregulatory, chemoprotective, cardioprotective, and recovery from neurodegenerative disorders. With the growing realization of benefits and associated challenges in the improvement of W. somnifera, studies on exploration of genetic and chemotypic variations, identification and characterization of important genes, and understanding the secondary metabolites production and their modulation has gained significant momentum. In recent years, several in vitro and in vivo preclinical studies have facilitated the validation of therapeutic potential of the phytochemicals derived from W. somnifera and have provided necessary impetus for gaining deeper insight into the mechanistic aspects involved in the mode of action of these important pharmaceutically active constituents. The present review highlights some of the current developments and future prospects of biotechnological intervention in this important medicinal plant.

Heterogeneous nuclear ribonucleoprotein K (hnRNP-K) promotes tumor metastasis by induction of genes involved in extracellular matrix, cell movement, and angiogenesis.

Cancer is a leading cause of death and still awaits effective therapies. Rapid industrialization has contributed to increase in incidence of cancer. One of the reasons why most of the cancers fail therapy is due to their metastatic property. Hence identification of factors leading to metastasis is highly important to design effective and novel anti-cancer therapeutics. In our earlier study (Inoue, A., Sawata, S. Y., Taira, K., and Wadhwa, R. (2007) Loss-of-function screening by randomized intracellular antibodies: identification of hnRNP-K as a potential target for metastasis. Proc. Natl. Acad. Sci. U.S.A. 104, 8983-8988), we had reported that the involvement of heterogeneous nuclear ribonucleoprotein K (hnRNP-K) in metastasis. Here, we established hnRNP-K-overexpressing and -underexpressing derivative cell lines and examined their proliferation and metastatic properties in vitro and in vivo. Whereas hnRNP-K compromised cells showed delayed tumor growth, its overexpression resulted in enhanced malignancy and metastasis. Molecular basis of the hnRNP-K induced malignant and metastatic phenotypes was dissected by cDNA microarray and pathway analyses. We found that the hnRNP-K regulates extracellular matrix, cell motility, and angiogenesis pathways. Involvement of the selected genes (Cck, Mmp-3, Ptgs2, and Ctgf) and pathways was validated by gene-specific expression analysis. Our results demonstrated that the hnRNP-K is a potential target for metastasis therapy.

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.

Mortalin antibody-conjugated quantum dot transfer from human mesenchymal stromal cells to breast cancer cells requires cell-cell interaction.

The role of tumor stroma in regulation of breast cancer growth has been widely studied. However, the details on the type of heterocellular cross-talk between stromal and breast cancer cells (BCCs) are still poorly known. In the present study, in order to investigate the intercellular communication between human mesenchymal stromal cells (hMSCs) and breast cancer cells (BCCs, MDA-MB-231), we recruited cell-internalizing quantum dots (i-QD) generated by conjugation of cell-internalizing anti-mortalin antibody and quantum dots (QD). Co-culture of illuminated and color-coded hMSCs (QD655) and BCCs (QD585) revealed the intercellular transfer of QD655 signal from hMSCs to BCCs. The amount of QD double positive BCCs increased gradually within 48h of co-culture. We found prominent intercellular transfer of QD655 in hanging drop co-culture system and it was non-existent when hMSCs and BBCs cells were co-cultured in trans-well system lacking imminent cell-cell contact. Fluorescent and electron microscope analyses also supported that the direct cell-to-cell interactions may be required for the intercellular transfer of QD655 from hMSCs to BCCs. To the best of our knowledge, the study provides a first demonstration of transcellular crosstalk between stromal cells and BCCs that involve direct contact and may also include a transfer of mortalin, an anti-apoptotic and growth-promoting factor enriched in cancer cells.

Natural Compounds that Modulate Circadian Rhythms.

Circadian rhythms of innate 24 h cycles comprise well-conserved biological phenomena from cyanobacteria to mammalian. They are driven by light and regulated by clock genes that work as transcription factors and control the expression of many other genes and physiological functions in the cells. The expression of ~ 40% of protein-coding genes shows 24 h oscillation patterns in mice, implying their importance in normal body functions. Indeed, the physiological and behavioural rhythmicity generated through clock genes-mediated multiple mechanisms affects the quality of life at large. Disrupted circadian rhythmicity is associated with several kinds of diseases. For example, cancer cells show abnormal expression patterns for circadian rhythm genes that have been shown to regulate oncogenesis, drug responses, and disease prognosis. Furthermore, the modern globalisation of human lifestyle and business and social activities have disrupted innate circadian rhythm, resulting in a variety of diseases through disrupted humoral, immunological, and neuronal pathways. Safe and sustainable modulation of circadian rhythm has become a prevalent need that warrants basic and interventional research, as well as clinical investigations. Although traditional systems of medicine suggest some natural compounds with circadian rhythmmodulating potential, most of these have not been validated in laboratory or clinical studies. Reliable read-outs of the effects of test compounds on circadian rhythmicity have been limited by the availability of live cell assays. We have, herein, provided an overview of living cell-embedded real- time reporter gene assays designed for screening compounds that modulate circadian rhythm, and discussed the potential of some natural compounds for circadian rhythm modulation as validated by cell-based assay systems, and their role in disease therapeutics.