Inhibition of Human Colorectal Cancer by a Natural Product 7-Acetylhorminone and Interactions with BSA/HSA: Multispectral Analysis and In Silico and In Vitro Studies.

We have semi-synthesized a natural product 7-acetylhorminone from crude extract of Premna obtusifolia (Indian headache tree), which is active against colorectal cancer after probation through computational screening methods as it passed through the set parameters of pharmacokinetics (most important nonblood-brain barrier permeant) and drug likeliness (e.g., Lipinski's, Ghose's, Veber's rule) which most other phytoconstituents failed to pass combined with docking with EGFR protein which is highly upregulated in the colorectal carcinoma cell. The structure of 7-acetylhorminone was confirmed by single crystal X-ray diffraction studies and 1H NMR, 13C NMR, and COSY studies. To validate the theoretical studies, first, in vitro experiments were carried out against human colorectal carcinoma cell lines (HCT116) which revealed the potent cytotoxic efficacy of 7-acetylhorminone and verified preliminary investigation. Second, the drugability of 7-acetylhorminone interaction with serum albumin proteins (HSA and BSA) is evaluated both theoretically and experimentally via steady-state fluorescence spectroscopic studies, circular dichroism, isothermal titration calorimetry, and molecular docking. In summary, this study reveals the applicability of 7-acetylhorminone as a potent drug candidate or as a combinatorial drug against colorectal cancer.

Divergent mutations of Delta and Omicron variants: key players behind differential viral attributes across the COVID-19 waves.

The third SARS-CoV-2 pandemic wave causing Omicron variant has comparatively higher replication rate and transmissibility than the second wave-causing Delta variant. The exact mechanism behind the differential properties of Delta and Omicron in respect to infectivity and virulence is not properly understood yet. This study reports the analysis of different mutations within the receptor binding domain (RBD) of spike glycoprotein and non-structural protein (nsp) of Delta and Omicron strains. We have used computational studies to evaluate the properties of Delta and Omicron variants in this work. Q498R, Q493R and S375F mutations of RBD showed better docking scores for Omicron compared to Delta variant of SARS-CoV-2, whereas nsp3_L1266I with PARP15 (7OUX), nsp3_L1266I with PARP15 (7OUX), and nsp6_G107 with ISG15 (1Z2M) showed significantly higher docking score. The findings of the present study might be helpful to reveal the probable cause of relatively milder form of COVID-19 disease manifested by Omicron in comparison to Delta variant of SARS-CoV-2 virus. Supplementary Information: The online version contains supplementary material available at 10.1007/s13337-023-00823-0.

Design of a Structurally Novel Multipotent Drug Candidate by the Scaffold Architecture Technique for ACE-II, NSP15, and Mpro Protein Inhibition: Identification and Isolation of a Natural Product to Prevent the Severity of Future Variants of Covid 19 and a Colorectal Anticancer Drug.

Scaffold architecture in the sectors of biotechnology and drug discovery research include scaffold hopping and molecular modelling techniques and helps in searching for potential drug candidates containing different core structures using computer-based software, which greatly aids medicinal and pharmaceutical chemistry. Going ahead, the computational method of scaffold architecture is thought to produce new scaffolds, and the method is capable of helping search engines toward producing new scaffolds that are likely to represent potent compounds with high therapeutic applications, which is a possibility in this case as well. Here we probate a different interactive design by natural product hopping, molecular modelling, pharmacophore modelling, modification, and combination of the phytoconstituents present in different medicinal plants for developing a pharmacophore-guided good drug candidate for the variants of SARS-CoV-2 or Covid 19. In the modern era, these approaches are carried out at every level of development of scaffold queries, which are increasingly summarized from chemical structures. In this context, we report on a successfully designed drug-like candidate having a high-binding-affinity "compound SLP" by understanding the relationships between the compounds' pharmacophores, scaffold functional groups, and biological activities beyond their individual applications that abide by Lipinski's rule of five, Ghose rule, Veber rule etc. The new scaffold generated by altering the core of the known phyto-compounds holds a good predicted ADMET profile and is examined with iMODS server to check the molecular dynamics simulation with normal mode analysis (NMA). The scaffold's three-dimensional (3D) structure yields a searchable natural product koenimbine from a conformer database having good ADMET property and high availability in spice Murraya koenigii leaves. M. koenigii leaves are easily available in the market, and might ensure the immunity, good health, and well-being of people if affected with any of the variants of Covid 19. The cell viability studies of koenimbine on murine colorectal carcinoma cell line (CT-26) showed no toxicity on normal mice lymphocyte cells (MLCs). The anticancer mechanism of koenimbine was displayed by its enhanced capacity to produce intercellular reactive oxygen species (ROS) in the colorectal carcinoma cell line.

Short Amphiphiles or Micelle Peptides May Help to Fight Against COVID-19.

BACKGROUND: COVID-19 is a global threat as a result of the incessant spread of SARS-CoV- 2, necessitating the rapid availability of effective antiviral medications to protect our society. For SARSCoV- 2, a group of peptides has already been indicated, although their effectiveness has yet to be shown. SARS-CoV-2 is an enveloped virus with hydrophobic fusion protein and spike glycoproteins. METHODS: Here, we have compiled a list of amphiphilic peptides that have been published, as well as their in-silico docking studies with the SARS-CoV-2 spike glycoprotein. RESULTS: The findings demonstrated that spike protein and amphiphilic peptides with increased binding affinity create a complex. It was also observed that PalL1 (ARLPRTMVHPKPAQP), 10AN1 (FWFTLIKTQAKQPARYRRFC), THETA defensin (RCICGRGICRLL), and mucroporin M1 (LFRLIKSLIKRLVSAFK) showed the binding free energy of more than -1000 kcal/mol. Molecular pI and hydrophobicity are also important factors of peptides to enhance the binding affinity with spike protein of SARS-CoV-2. CONCLUSION: In light of these findings, it is crucial to compare the in-vitro to in-vivo efficacy of amphiphilic peptides in order to produce an efficient anti-SARS-CoV-2 peptide therapy that might assist control the present pandemic scenario.

Facile and Green Fabrication of Highly Competent Surface-Modified Chlorogenic Acid Silver Nanoparticles: Characterization and Antioxidant and Cancer Chemopreventive Potential.

The eco-friendly, cost-effective, and green fabrication of nanoparticles is considered a promising area of nanotechnology. Here, we report on the green synthesis and characterization of bovine serum albumin (BSA)-decorated chlorogenic acid silver nanoparticles (AgNPs-CGA-BSA) and the studies undertaken to verify their plausible antioxidant and antineoplastic effects. High-resolution transmission electron microscopy (HR-TEM), dynamic light scattering, X-ray diffraction, and Fourier transform infrared analyses depict an average mean particle size of ∼96 nm, spherical morphology, and nanocrystalline structure of AgNPs-CGA-BSA. DPPH scavenging and inhibition of lipid peroxidation signify the noticeable in vitro antioxidant potential of the nanoparticles. The in vitro experimental results demonstrate that AgNPs-CGA-BSA shows significant cytotoxicity to Dalton's lymphoma ascites (DLA) cells and generates an enhanced intracellular reactive oxygen species and oxidized glutathione (GSSG) and reduced glutathione (GSH) in DLA cells. Furthermore, mechanism investigation divulges the pivotal role of the downregulated expression of superoxide dismutase (SOD) and catalase (CAT), and these ultimately lead to apoptotic chromatin condensation in AgNPs-CGA-BSA-treated DLA cells. In addition, in vivo experiments reveal an excellent decrease in tumor cell count, an increase in serum GSH and CAT, SOD, and glutathione peroxidase activities, and a decrease in the malondialdehyde (MDA) level in DLA-bearing mice after AgNPs-CGA-BSA treatment. These findings suggest that the newly synthesized biogenic green silver nanoparticles have remarkable in vitro antioxidant and antineoplastic efficacy that triggers cytotoxicity, oxidative stress, and chromatin condensation in DLA cells and in vivo anticancer efficacy that enhances the host antioxidant status, and these might open a new path in T-cell lymphoma therapy.

Bioinformatics and Network Pharmacology of the First Crystal Structured Clerodin: Anticancer and Antioxidant Potential against Human Breast Carcinoma Cell.

Clerodin was isolated from the medicinal plant Clerodendrum infortunatum, and CSD search showed the first crystal structure of clerodin by a single-crystal X-ray diffraction study. We checked its binding potential with target proteins by docking and conducted network pharmacology analysis, ADMET analysis, in silico pathway analysis, normal mode analysis (NMA), and cytotoxic activity studies to evaluate clerodin as a potential anticancer agent. The cell viability studies of clerodin on the human breast carcinoma cell line (MCF-7) showed toxicity on MCF-7 cells but no toxicity toward normal human lymphocyte cells (HLCs). The anticancer mechanism of clerodin was validated by its enhanced capacity to produce intracellular reactive oxygen species (ROS) and to lower the reduced glutathione content in MCF-7 cells.

Efficient Propagation of Circulating Tumor Cells: A First Step for Probing Tumor Metastasis.

Circulating tumor cells (CTCs) represent a unique population of cells that can be used to investigate the mechanistic underpinnings of metastasis. Unfortunately, current technologies designed for the isolation and capture of CTCs are inefficient. Existing literature for in vitro CTC cultures report low (6-20%) success rates. Here, we describe a new method for the isolation and culture of CTCs. Once optimized, we employed the method on 12 individual metastatic breast cancer patients and successfully established CTC cultures from all 12 samples. We demonstrate that cells propagated were of breast and epithelial origin. RNA-sequencing and pathway analysis demonstrated that CTC cultures were distinct from cells obtained from healthy donors. Finally, we observed that CTC cultures that were associated with CD45+ leukocytes demonstrated higher viability. The presence of CD45+ leukocytes significantly enhanced culture survival and suggests a re-evaluation of the methods for CTC isolation and propagation. Routine access to CTCs is a valuable resource for identifying genetic and molecular markers of metastasis, personalizing the treatment of metastatic cancer patients and developing new therapeutics to selectively target metastatic cells.

Conditional Reprogramming for Patient-Derived Cancer Models and Next-Generation Living Biobanks.

Traditional cancer models including cell lines and animal models have limited applications in both basic and clinical cancer research. Genomics-based precision oncology only help 2-20% patients with solid cancer. Functional diagnostics and patient-derived cancer models are needed for precision cancer biology. In this review, we will summarize applications of conditional cell reprogramming (CR) in cancer research and next generation living biobanks (NGLB). Together with organoids, CR has been cited in two NCI (National Cancer Institute, USA) programs (PDMR: patient-derived cancer model repository; HCMI: human cancer model initiatives. HCMI will be distributed through ATCC). Briefly, the CR method is a simple co-culture technology with a Rho kinase inhibitor, Y-27632, in combination with fibroblast feeder cells, which allows us to rapidly expand both normal and malignant epithelial cells from diverse anatomic sites and mammalian species and does not require transfection with exogenous viral or cellular genes. Establishment of CR cells from both normal and tumor tissue is highly efficient. The robust nature of the technique is exemplified by the ability to produce 2 × 106 cells in five days from a core biopsy of tumor tissue. Normal CR cell cultures retain a normal karyotype and differentiation potential and CR cells derived from tumors retain their tumorigenic phenotype. CR also allows us to enrich cancer cells from urine (for bladder cancer), blood (for prostate cancer), and pleural effusion (for non-small cell lung carcinoma). The ability to produce inexhaustible cell populations using CR technology from small biopsies and cryopreserved specimens has the potential to transform biobanking repositories (NGLB: next-generation living biobank) and current pathology practice by enabling genetic, biochemical, metabolomic, proteomic, and biological assays, including chemosensitivity testing as a functional diagnostics tool for precision cancer medicine. We discussed analyses of patient-derived matched normal and tumor models using a case with tongue squamous cell carcinoma as an example. Last, we summarized applications in cancer research, disease modeling, drug discovery, and regenerative medicine of CR-based NGLB.

Rhodamine-azobenzene based single molecular probe for multiple ions sensing: Cu2+, Al3+, Cr3+ and its imaging in human lymphocyte cells.

A photoinduced electron transfer (PET) and chelation-enhanced fluorescence (CHEF) regulated rhodamine-azobenzene chemosensor (L) was synthesized for chemoselective detection of Al3+, Cr3+, and Cu2+ by UV-Visible absorption study whereas Al3+ and Cr3+ by fluorimetric study in EtOH-H2O solvent. L showed a clear fluorescence emission enhancement of 21 and 16 fold upon addition of Al3+ and Cr3+ due to the 1:1 host-guest complexation, respectively. This is first report on rhodamine-azobenzene based Cr3+ chemosensor. The complex formation, restricted imine isomerization, inhibition of PET (photo-induced electron transfer) process with the concomitant opening of the spirolactam ring induced a turn-on fluorescence response. The higher binding constants 6.7 × 103 M-1 and 3.8 × 103 M-1 for Al3+ and Cr3+, respectively and lower detection limits 1 × 10-6 M and 2 × 10-6 M for Al3+ and Cr3+, respectively in a buffered solution with high reversible nature describes the potential of L as an effective tool for detecting Al3+ and Cr3+ in a biological system with higher intracellular resolution. Finally, L was used to map the intracellular concentration of Al3+ and Cr3+ in human lymphocyte cells (HLCs) at physiological pH very effectively. Altogether, our findings will pave the way for designing new chemosensors for multiple analytes and those chemosensors will be effective for cell imaging study.

Antigonadal and endocrine-disrupting activities of lambda cyhalothrin in female rats and its attenuation by taurine.

Lambda cyhalothrin (LCT) is a type II pyrethroid with a wide range of agricultural, industrial, and household uses. Taurine is a nonprotein sulfur containing amino acid as well as a well-known antioxidant and has valuable clinical applications in the detoxification of xenobiotics. The present study evaluated the effect of LCT on the reproductive and endocrine systems of female rats and determined whether taurine might alter these effects. Sexually mature female rats were administered LCT at two different dosages (6.3 mg/kg BW and 11.33 mg/kg BW) once daily by oral gavage for 14 consecutive days with the pretreatment of taurine (50 mg kg-1 BW). LCT treatment resulted in diminished adrenal cholesterol, ovarian 3ß- and 17ß-hydroxysteroid dehydrogenase (HSD) activity with increased ovarian cholesterol, adrenal 3ß- and 17ß-HSD activity. Furthermore, protein and mRNA expressions of ovarian 17ß-HSD and steroidogenic acute regulatory protein were also decreased. Hormonal imbalance was evident by concurrent reduction in the gonadotropic hormone, estradiol, and progesterone levels in LCT-treated rats. These rats also demonstrated the histopathological evidence of degenerative changes in the ovaries. Pretreatment of taurine attenuated the LCT-induced changes.

Synthesis and characterization of biogenic metal nanoparticles and its cytotoxicity and anti-neoplasticity through the induction of oxidative stress, mitochondrial dysfunction and apoptosis.

In the present study, we demonstrate a simple, cost-effective and eco-friendly method for biogenic synthesis of silver nanoparticles (AgNPCGs) using ethanolic extract of Calotropis gigantea latex. Attempts were made to characterize these biogenic silver nanoparticles AgNPCGs and also to test its cytotoxic, anti-neoplastic and apoptotic potential through the induction of oxidative stress, mitochondrial dysfunction. AgNPCGs were characterized by UV-vis spectroscopy, dynamic light scattering (DLS) and surface zeta potential measurement, Fourier transform infrared (FTIR) spectroscopy, X-ray diffraction (XRD), transmission electron microscopy (TEM) and selected area electron diffraction, scanning electron microscopy (SEM), energy-dispersive X-ray fluorescence spectrometry (EDX). UV visible spectroscopy showed an intense surface plasmon resonance band at 431nm which clearly reflected the formation of silver nanoparticles. FTIR study revealed that latex extract acted as reducing and stabilizing agent for the synthesis of AgNPCGs. Energy dispersive X-ray spectroscopy confirmed the presence of silver as a major component of synthesized AgNPCGs. SEM and TEM studies showed that the synthesized AgNPCGs were nearly spherical in shape with an average size of 2.338nm. The selected area electron diffraction pattern and XRD studies confirmed the crystalline nature of AgNPCGs. AgNPCGs exhibited in-vitro cytotoxic activity against Ehrlich's ascites carcinoma (EAC), Jurkat and MCF-7 cells at respective IC50 doses without producing cytotoxicity to mice and human lymphocytes. Significant chromatin condensation, DNA fragmentation, cell cycle arrest at G2/M phase, up-regulation of Bax and caspase-3 and down-regulation of Bcl-2 were observed in AgNPCGs treated EAC cells. The results suggest that biogenic silver nanoparticles AgNPCGs could be a potential chemotherapeutic formulation for cancer therapy.

A multiplex preclinical model for adenoid cystic carcinoma of the salivary gland identifies regorafenib as a potential therapeutic drug.

Adenoid cystic carcinomas (ACC) are rare salivary gland cancers with a high incidence of metastases. In order to study this tumor type, a reliable model system exhibiting the molecular features of this tumor is critical, but none exists, thereby inhibiting in-vitro studies and the analysis of metastatic behavior. To address this deficiency, we have coupled an efficient method to establish tumor cell cultures, conditional reprogramming (CR), with a rapid, reproducible and robust in-vivo zebrafish model. We have established cell cultures from two individual ACC PDX tumors that maintain the characteristic MYB translocation. Additional mutations found in one ACC culture also seen in the PDX tumor. Finally, the CR/zebrafish model mirrors the PDX mouse model and identifies regorafenib as a potential therapeutic drug to treat this cancer type that mimic the drug sensitivity profile in PDX model, further confirming the unique advantages of multiplex system.

Conditional cell reprogramming involves non-canonical ß-catenin activation and mTOR-mediated inactivation of Akt.

The combination of irradiated fibroblast feeder cells and Rho kinase inhibitor, Y-267362, converts primary epithelial cells growing in vitro into an undifferentiated adult stem cell-like state that is characterized by long-term proliferation. This cell culture method also maintains the proliferation of adult epithelial stem cells from various tissues. Both primary and adult stem cells retain their tissue-specific differentiation potential upon removal of the culture conditions. Due to the ability to modulate the proliferation and differentiation of the cells, this method is referred to as conditional reprogramming and it is increasingly being used in studies of tumor heterogeneity, personalized medicine and regenerative medicine. However, little is known about the biology of these conditionally reprogrammed (CR) cells. Previously we showed that ß-catenin activation, a hallmark of stem cells in vivo, occurs in CR human ectocervical cells (HECs). Here we show that ß-catenin-dependent transcription is necessary for the induction of epithelial stem cell markers, and that ß-catenin is activated via a non-canonical pathway that is independent of Wnt and Akt/GSK-3. Active Akt actually decreases due to increased mTOR signaling, with a consequent increase in dephosphorylated, active GSK-3. Despite the increase in active GSK-3, ß-catenin associates with protein phosphatase 2A (PP2A) and is activated. Inhibition of PP2A catalytic activity reduces both the level of active ß-catenin and the acute induction of stem cell markers, suggesting an important role for PP2A in the activation of ß-catenin. Moreover, we demonstrate similar results using human prostate and breast cells, indicating that these changes are not restricted to ectocervical epithelial cells and may represent a more fundamental property of conditional reprogramming.

A new sub-pathway of long-patch base excision repair involving 5' gap formation.

Base excision repair (BER) is one of the most frequently used cellular DNA repair mechanisms and modulates many human pathophysiological conditions related to DNA damage. Through live cell and in vitro reconstitution experiments, we have discovered a major sub-pathway of conventional long-patch BER that involves formation of a 9-nucleotide gap 5' to the lesion. This new sub-pathway is mediated by RECQ1 DNA helicase and ERCC1-XPF endonuclease in cooperation with PARP1 poly(ADP-ribose) polymerase and RPA The novel gap formation step is employed during repair of a variety of DNA lesions, including oxidative and alkylation damage. Moreover, RECQ1 regulates PARP1 auto-(ADP-ribosyl)ation and the choice between long-patch and single-nucleotide BER, thereby modulating cellular sensitivity to DNA damage. Based on these results, we propose a revised model of long-patch BER and a new key regulation point for pathway choice in BER.

Conditional reprogramming and long-term expansion of normal and tumor cells from human biospecimens.

Historically, it has been difficult to propagate cells in vitro that are derived directly from human tumors or healthy tissue. However, in vitro preclinical models are essential tools for both the study of basic cancer biology and the promotion of translational research, including drug discovery and drug target identification. This protocol describes conditional reprogramming (CR), which involves coculture of irradiated mouse fibroblast feeder cells with normal and tumor human epithelial cells in the presence of a Rho kinase inhibitor (Y-27632). CR cells can be used for various applications, including regenerative medicine, drug sensitivity testing, gene expression profiling and xenograft studies. The method requires a pathologist to differentiate healthy tissue from tumor tissue, and basic tissue culture skills. The protocol can be used with cells derived from both fresh and cryopreserved tissue samples. As approximately 1 million cells can be generated in 7 d, the technique is directly applicable to diagnostic and predictive medicine. Moreover, the epithelial cells can be propagated indefinitely in vitro, yet retain the capacity to become fully differentiated when placed into conditions that mimic their natural environment.

Conditionally reprogrammed normal and primary tumor prostate epithelial cells: a novel patient-derived cell model for studies of human prostate cancer.

Our previous study demonstrated that conditional reprogramming (CR) allows the establishment of patient-derived normal and tumor epithelial cell cultures from a variety of tissue types including breast, lung, colon and prostate. Using CR, we have established matched normal and tumor cultures, GUMC-29 and GUMC-30 respectively, from a patient's prostatectomy specimen. These CR cells proliferate indefinitely in vitro and retain stable karyotypes. Most importantly, only tumor-derived CR cells (GUMC-30) produced tumors in xenografted SCID mice, demonstrating maintenance of the critical tumor phenotype. Characterization of cells with DNA fingerprinting demonstrated identical patterns in normal and tumor CR cells as well as in xenografted tumors. By flow cytometry, both normal and tumor CR cells expressed basal, luminal, and stem cell markers, with the majority of the normal and tumor CR cells expressing prostate basal cell markers, CD44 and Trop2, as well as luminal marker, CD13, suggesting a transit-amplifying phenotype. Consistent with this phenotype, real time RT-PCR analyses demonstrated that CR cells predominantly expressed high levels of basal cell markers (KRT5, KRT14 and p63), and low levels of luminal markers. When the CR tumor cells were injected into SCID mice, the expression of luminal markers (AR, NKX3.1) increased significantly, while basal cell markers dramatically decreased. These data suggest that CR cells maintain high levels of proliferation and low levels of differentiation in the presence of feeder cells and ROCK inhibitor, but undergo differentiation once injected into SCID mice. Genomic analyses, including SNP and INDEL, identified genes mutated in tumor cells, including components of apoptosis, cell attachment, and hypoxia pathways. The use of matched patient-derived cells provides a unique in vitro model for studies of early prostate cancer.

ROCK inhibitor reduces Myc-induced apoptosis and mediates immortalization of human keratinocytes.

The Myc/Max/Mad network plays a critical role in cell proliferation, differentiation and apoptosis and c-Myc is overexpressed in many cancers, including HPV-positive cervical cancer cell lines. Despite the tolerance of cervical cancer keratinocytes to high Myc expression, we found that the solitary transduction of the Myc gene into primary cervical and foreskin keratinocytes induced rapid cell death. These findings suggested that the anti-apoptotic activity of E7 in cervical cancer cells might be responsible for negating the apoptotic activity of over-expressed Myc. Indeed, our earlier in vitro studies demonstrated that Myc and E7 synergize in the immortalization of keratinocytes. Since we previously postulated that E7 and the ROCK inhibitor, Y-27632, were members of the same functional pathway in cell immortalization, we tested whether Y-27632 would inhibit apoptosis induced by the over-expression of Myc. Our findings indicate that Y-27632 rapidly inhibited Myc-induced membrane blebbing and cellular apoptosis and, more generally, functioned as an inhibitor of extrinsic and intrinsic pathways of cell death. Most important, Y-27632 cooperated with Myc to immortalize keratinocytes efficiently, indicating that apoptosis is a major barrier to Myc-induced immortalization of keratinocytes. The anti-apoptotic activity of Y-27632 correlated with a reduction in p53 serine 15 phosphorylation and the consequent reduction in the expression of downstream target genes p21 and DAPK1, two genes involved in the induction of cell death.

A novel method for monitoring functional lesion-specific recruitment of repair proteins in live cells.

DNA-protein relationships have been studied by numerous methods, but a particular gap in methodology lies in the study of DNA adduct-specific interactions with proteins in vivo, which particularly affects the field of DNA repair. Using the repair of a well-characterized and ubiquitous adduct, the abasic (AP) site, as a model, we have developed a comprehensive method of monitoring DNA lesion-specific recruitment of proteins in vivo over time. We utilized a surrogate system in which a Cy3-labeled plasmid containing a single AP-site was transfected into cells, and the interaction of the labeled DNA with BER enzymes, including APE1, Polß, LIG1, and FEN1, was monitored by immunofluorescent staining of the enzymes by Alexafluor-488-conjugated secondary antibody. The recruitment of enzymes was characterized by quantification of Cy3-Alexafluor-488 co-localization. To validate the microscopy-based method, repair of the transfected AP-site DNA was also quantified at various time points post-transfection using a real time PCR-based method. Notably, the recruitment time kinetics for each enzyme were consistent with AP-site repair time kinetics. This microscopy-based methodology is reliable in detecting the recruitment of proteins to specific DNA substrates and can be extended to study other in vivo DNA-protein relationships in any DNA sequence and in the context of any DNA structure in transfectable proliferating or quiescent cells. The method may be applied to a variety of disciplines of nucleic acid transaction pathways, including repair, replication, transcription, and recombination.

Repair kinetics of acrolein- and (E)-4-hydroxy-2-nonenal-derived DNA adducts in human colon cell extracts.

ω-3 and ω-6 polyunsaturated fatty acids (PUFAs) play a role in the pathogenesis of colon cancer. Upon oxidation, PUFAs generate α,ß-unsaturated aldehydes or enals, such as acrolein (Acr) and (E)-4-hydroxy-2-nonenal (HNE), which can form cyclic adducts of deoxyguanosine (Acr-dG and HNE-dG, respectively) in DNA. Both Acr-dG and HNE-dG adducts have been detected in human and animal tissues and are potentially mutagenic and carcinogenic. In vivo levels of Acr-dG in DNA are at least two orders of magnitude higher than those of HNE-dG. In addition to the facile reaction with Acr, the higher levels of Acr-dG than HNE-dG in vivo may be due to a lower rate of repair. Previous studies have shown that HNE-dG adducts are repaired by the NER pathway (Choudhury et al. [42]). We hypothesize that Acr-dG adducts are repaired at a slower rate than HNE-dG and that HNE-dG in DNA may influence the repair of Acr-dG. In this study, using a DNA repair synthesis assay and a LC-MS/MS method, we showed that Acr-dG in a plasmid DNA is repaired by NER proteins, but it is repaired at a much slower rate than HNE-dG in human colon cell extracts, and the slow repair of Acr-dG is likely due to poor recognition/excision of the lesions in DNA. Furthermore, using a plasmid DNA containing both adducts we found the repair of Acr-dG is significantly inhibited by HNE-dG, however, the repair of HNE-dG is not much affected by Acr-dG. This study demonstrates that the NER repair efficiencies of the two major structurally-related in vivo cyclic DNA adducts from lipid oxidation vary greatly. More importantly, the repair of Acr-dG can be significantly retarded by the presence of HNE-dG in DNA. Therefore, this study provides a mechanistic explanation for the higher levels of Acr-dG than HNE-dG observed in tissue DNA.

Dissecting the pathways that destabilize mutant p53: the proteasome or autophagy?

One fundamental feature of mutant forms of p53 consists in their accumulation at high levels in tumors. At least in the case of neomorphic p53 mutations, which acquire oncogenic activity, stabilization is a driving force for tumor progression. It is well documented that p53 mutants are resistant to proteasome-dependent degradation compared with wild-type p53, but the exact identity of the pathways that affect mutant p53 stability is still debated. We have recently shown that macroautophagy (autophagy) provides a route for p53 mutant degradation during restriction of glucose. Here we further show that in basal conditions of growth, inhibition of autophagy with chemical inhibitors or by downregulation of the essential autophagic genes ATG1/Ulk1, Beclin-1 or ATG5, results in p53 mutant stabilization. Conversely, overexpression of Beclin-1 or ATG1/Ulk1 leads to p53 mutant depletion. Furthermore, we found that in many cell lines, prolonged inhibition of the proteasome does not stabilize mutant p53 but leads to its autophagic-mediated degradation. Therefore, we conclude that autophagy is a key mechanism for regulating the stability of several p53 mutants. We discuss plausible mechanisms involved in this newly identified degradation pathway as well as the possible role played by autophagy during tumor evolution driven by mutant p53.