Search

Mutations in the tumor suppressor protein p53 is a prevalent feature in majority of cancers resulting in inactivation of its activities related to control of cell cycle progression and proliferation. p53 Y220C is one of the common hotspot mutations that causes decrease in its thermodynamic stability. Some small molecules have been shown to bind to the mutated site and restore its wild type thermodynamics and tumor suppressor function. In this study, we have explored the potential of caffeic acid phenethyl ester (CAPE-a bioactive compound from propolis) to interact with p53 Y220C and restore its wild type p53 (p53 wt ) transcription activation and tumor suppressor activities. We recruited computational methods, viz. molecular docking, molecular dynamics simulations and free energy calculations to study the interaction of CAPE at the mutation crevice and found that it has potential to restore p53 wt function of the p53 Y220C mutant similar to a previously described restoration molecule PK7242. We provide cell-based experimental evidence to these predictions and suggest CAPE as a potential natural drug for treatment of p53 Y220C mutant harboring cancers., (© 2021. The Author(s).)

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.

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.

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.

Mortalin is a stress chaperone belonging to the Hsp70 family of proteins. Frequently enriched in cancers, it is a multifunctional protein and regulates cell proliferation, apoptosis, mitochondrial functions, and the activity of tumor suppressor protein p53. In the present study, we investigated circulating mortalin and its autoantibody in normal, cirrhosis, and cancerous liver. We found that although mortalin is enriched in liver cancer cells and tumors, it is not detected in the serum of either the liver cirrhosis or cancer patients. In contrast, mortalin autoantibody was detected in patients' sera and showed significant correlation with the occurrence of cirrhosis. It is suggested as a potential noninvasive marker for liver cirrhosis.

Cell therapy using human mesenchymal stem cells (MSCs) is an attractive approach for many refractory diseases. Adipose tissue-derived mesenchymal stem cells (AT-MSCs) are considered as a favorable tool due to its abundance in the body, easy proliferation, and high cytokine production potency. In order to avoid the risks associated with the use of fetal bovine serum (FBS) in culture that includes batch variations and contamination with pathogens, development of serum-free culture system has been initiated. We have formulated a completely serum-free culture medium (SFM) that could be used not only for the expansion of AT-MSCs but also for initial isolation. We demonstrate that the AT-MSCs isolated and cultured in serum-free medium (AT-MSCs/SFM) possess high proliferation capacity and differentiation potency to osteoblast, adipocyte, and chondrocyte lineages in vitro. In in vivo bone fraction model analysis, AT-MSCs/SFM showed higher bone repair potency and quality of the regenerated bone than the cells cultured in serum-containing medium (AT-MSCs/SCM). This was attributed to the (i) presence of translated cells in the bone, as evidenced by in vivo imaging of the illuminated translated cells and (ii) high level of expression and induction capacity of AT-MSCs/SFM for cytokine BMP2, CCL2, and CCL5. Taken together, we report a new serum-free culture system for AT-MSCs that is suitable for cell therapy.

The heat shock chaperones mortalin/mitochondrial heat shock protein 70 (mtHsp70) and Hsp60 are found in multiple subcellular sites and function in the folding and intracellular trafficking of many proteins. The chaperoning activity of these 2 proteins involves different structural and functional mechanisms. In spite of providing an excellent model for an evolutionarily conserved molecular "brotherhood", their individual functions, although overlapping, are nonredundant. As they travel to various locations, both chaperones acquire different binding partners and exert a more divergent involvement in tumorigenesis, cellular senescence, and immunology. An understanding of their functional biology may lead to novel designing and development of therapeutic strategies for cancer and aging.

Mortalin/mthsp70/PBP74/Grp75 (called mortalin hereafter), a member of the Hsp70 family of chaperones, was shown to have different subcellular localizations in normal and immortal cells. It has been assigned to multiple subcellular sites and implicated in multiple functions ranging from stress response, intracellular trafficking, antigen processing, control of cell proliferation, differentiation, and tumorigenesis. The present article compiles and reviews information on the multiple sites and functions of mortalin in different organisms. The relevance of its differential distributions and functions in normal and immortal cell phenotypes is discussed.

We have investigated the effects of acetone and methanol extracts of a medicinal plant, Terminalia arjuna, on the growth of human normal fibroblasts (WI-38), osteosarcoma (U2OS), and glioblastoma (U251) cells in vitro. We found that both extracts at 30 microg and 60 microg/ml concentrations inhibit the growth of transformed cells; the growth of normal cells was least affected. Although the transformed cells appeared to have fragmented nucleus by Hoechst staining, no deoxy-ribonucleic acid laddering effect was observed. In response to the extract treatment, the tumor suppressor protein, p53, was induced in U2OS but not in U251 and WI-38 cells. A cyclin-dependent kinase inhibitor, p21WAF1, was induced in transformed cells only. The study suggests that the bark extract of medicinal plant, T. arjuna, has components that can induce growth arrest of transformed cells by p53-dependent and -independent pathways.

A reverse Northern analysis that effectively eliminates the false positives isolated from differential display of mRNAs (DD) is demonstrated. Preparation of probe by one-step labeling in reverse transcription reaction is found to be more effective and specific as compared to the preparation of probe by random priming of reverse transcribed cDNA pool. Reverse Northern assay of DNA fragments isolated from DD prior to their cloning into plasmid, analysis of multiple fragments on single slot blot, and requirement of RNA only in small amounts as compared to conventional Northern makes the protocol quick, effective, and economic.