Repurposing of FDA approved kinase inhibitor bosutinib for mitigation of radiation induced damage via inhibition of JNK pathway.

Radiotherapy is a common modality for cancer treatment. However, it is often associated with normal tissue toxicity in 20-80% of the patients. Radioprotectors can improve the outcome of radiotherapy by selectively protecting normal cells against radiation toxicity. In the present study, compound libraries containing 54 kinase inhibitors and 80 FDA-approved drugs were screened for radioprotection of lymphocytes using high throughput cell analysis. A second-generation FDA-approved kinase inhibitor, bosutinib, was identified as a potential radioprotector for normal cells. The radioprotective efficacy of bosutinib was evinced from a reduction in radiation induced DNA damage, caspase-3 activation, DNA fragmentation and apoptosis. Oral administration of bosutinib protected mice against whole body irradiation (WBI) induced morbidity and mortality. Bosutinib also reduced radiation induced bone-marrow aplasia and hematopoietic damage in mice exposed to 4 Gy and 6 Gy dose of WBI. Mechanistic studies revealed that the radioprotective action of bosutinib involved interaction with cellular thiols and modulation of JNK pathway. The addition of glutathione and N-acetyl cysteine significantly reduced the radioprotective efficacy of bosutinib. Moreover, bosutinib did not protect cancer cells against radiation induced toxicity. On the contrary, bosutinib per se exhibited anticancer activity against human cancer cell lines. The results highlight possible use of bosutinib as a repurposable radioprotective agent for mitigation of radiation toxicity in cancer patients undergoing radiotherapy.

Mitochondriotropic Derivative of Ethyl Ferulate, a Dietary Phenylpropanoid, Exhibits Enhanced Cytotoxicity in Cancer Cells via Mitochondrial Superoxide-Mediated Activation of JNK and AKT Signalling.

Specific targeting of anti-cancer drugs to mitochondria is an emerging strategy to enhance cancer cell killing whilst simultaneously overcoming the problem of drug resistance, low bioavailability and limited clinical success of natural products. We have synthesized a mitochondria targeted derivative of Ethyl Ferulate (EF, a naturally occurring ester of ferulic acid), by conjugating it with triphenylphosphonium ion and compared its cytotoxicity with the parent molecule. Mito-Ethyl Ferulate (M-EF) was found to be more potent than EF (~ 400-fold) in inhibiting the growth of A549 and MCF-7 cells and suppressing the clonogenic potential of A549 cells. Notably, M-EF did not induce any cytotoxicity in normal cells (mouse normal fibroblast cells) up to a concentration of 25 µM. Furthermore, M-EF treatment induced significantly higher cell death in MCF-7 and A549 cells, as compared to EF via induction of apoptosis. M-EF treatment increased mitochondrial superoxide production and induced mitochondrial DNA damage and phosphorylation of JNK and AKT in A549 cells. Furthermore, M-EF induced increase in mitochondrial superoxide production and cytotoxicity was attenuated on pre-treatment with mitochondria-targeted antioxidant (mitoTEMPO) indicating the involvement of mitochondrial ROS in the cytotoxic effects of M-EF. Finally, in silico prediction revealed putative mitochondrial targets of M-EF which are known to regulate mitochondrial ROS and cell viability. In conclusion, the improved cytotoxic efficacy of M-EF exemplifies the use of mitochondria-specific drug delivery in future development of natural product based mitochondrial pharmacology.

Molecular dynamic simulations reveal anti-SARS-CoV-2 activity of mitocurcumin by potentially blocking innate immune evasion proteins NSP3 and NSP16.

The coronavirus disease 19 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is affecting human life in an unprecedented manner and has become a global public health emergency. Identification of novel inhibitors of viral infection/replication is the utmost priority to curtail COVID-19 progression. A pre-requisite for such inhibitors is good bioavailability, non-toxicity and serum stability. Computational studies have shown that curcumin can be a candidate inhibitor of certain SARS-CoV-2 proteins; however, poor bio-availability of curcumin limits its possible therapeutic application. To circumvent this limitation, we have used mitocurcumin (MC), a triphenyl phosphonium conjugated curcumin derivative, to study the ability to inhibit SARS-CoV-2 infection using molecular docking and molecular dynamics (MD) simulation. MC is serum stable and several fold more potent as compared to curcumin. Molecular docking studies revealed that MC can bind at active site of SARS-CoV-2 ADP Ribose Phosphatase (NSP3) and SARS-CoV-2 methyltransferase (NSP10-NSP16 complex) with a high binding energy of - 10.3 kcal/mol and - 10.4 kcal/mol, respectively. MD simulation (100 ns) studies revealed that binding of MC to NSP3 and NSP16 resulted in a stable complex. MC interacted with critical residues of NSP3 macro-domain and NSP10-NSP16 complex and occupied their active sites. NSP3 is known to suppress host immune responses whereas NSP10-NSP16 complex is known to prevent immune recognition of viral mRNA. Our study suggests that MC can potentially inhibit the activity of NSP3 and NSP10-NSP16 complex, resulting in compromised viral immune evasion mechanism, and thereby accentuate the innate immune mediated clearance of viral load.

Attention UW-Net: A fully connected model for automatic segmentation and annotation of chest X-ray.

BACKGROUND AND OBJECTIVE: Automatic segmentation and annotation of medical image plays a critical role in scientific research and the medical care community. Automatic segmentation and annotation not only increase the efficiency of clinical workflow, but also prevent overburdening of radiologists. The objective of this work is to improve the accuracy and give a probabilistic map for automatic annotation from small data set to reduce the use of tedious and prone to error manual annotations from chest X-rays. METHOD: In this paper, we have proposed an attention UW-Net, which introduces an intermediate layer acting as a bridge between the encoder and decoder pathways. The intermediate layer is a series of fully connected convolutional layers generated from the upsampling of the final encoder layer connected to the corresponding up sampled and down sampled blocks via skip-connections. The intermediate layer is further connected to the decoder pathway using a downsampling layer. RESULTS: The proposed attention UW-Net is giving a very good performance, achieving an average F1-score of 95.7%, 80.9%, 81.0% and 77.6% for lung (large), heart (medium), trachea (small), and collarbone (small) object segmentations, respectively. The attention UW-Net outperforms not only in comparison to U-Net and its variations but also with respect to other standard recent automatic and semi-automatic segmentation/annotation models. An ablation study was also performed to find the best suited high-performing architecture. CONCLUSION: The uniformity in prediction accuracy of segmentation masks for all kinds of segmentation masks (large, medium, and small lesions) makes this model best for automatic annotation of organs.

Role of protein S-Glutathionylation in cancer progression and development of resistance to anti-cancer drugs.

The survival, functioning and proliferation of mammalian cells are highly dependent on the cellular response and adaptation to changes in their redox environment. Cancer cells often live in an altered redox environment due to aberrant neo-vasculature, metabolic reprogramming and dysregulated proliferation. Thus, redox adaptations are critical for their survival. Glutathione plays an essential role in maintaining redox homeostasis inside the cells by binding to redox-sensitive cysteine residues in proteins by a process called S-glutathionylation. S-Glutathionylation not only protects the labile cysteine residues from oxidation, but also serves as a sensor of redox status, and acts as a signal for stimulation of downstream processes and adaptive responses to ensure redox equilibrium. The present review aims to provide an updated overview of the role of the unique redox adaptations during carcinogenesis and cancer progression, focusing on their dependence on S-glutathionylation of specific redox-sensitive proteins involved in a wide range of processes including signalling, transcription, structural maintenance, mitochondrial functions, apoptosis and protein recycling. We also provide insights into the role of S-glutathionylation in the development of resistance to chemotherapy. Finally, we provide a strong rationale for the development of redox targeting drugs for treatment of refractory/resistant cancers.

Redox regulation of regulatory T-cell differentiation and functions.

The choice between immunity or tolerance is a consequence of T-cell fate determined by T-cell receptor affinity to cognate MHC-peptide complex, costimulatory molecules and cytokines from antigen presenting cells. While activated, effector and memory T-cells provide immunity against antigens, regulatory T-cells play a pivotal non-redundant role in immune tolerance and tissue repair. T-cell differentiation and functions are also well known to be governed by the redox status. Physiological redox status is determined by oxygen concentration, reactive oxygen species levels and antioxidant concentration (vitamin C, glutathione, vitamin E). Cellular redox state influences the levels of oxygen-dependent ten-eleven translocase (TET) demethylase, hypoxia inducible factor-1α (HIF-1α), and metabolic reprogramming which in turn control the epigenetic modification, transcription, translation and post-translational stability of FoxP3, the master regulator of regulatory T-cell induction and maintenance. Redox changes during foetal development, pregnancy, ageing, infections and cancer bolster Treg differentiation for immune tolerance to non-dangerous non-self-antigens. Incidentally, the changes in blood oxygen levels in pregnant women and developing foetus are accompanied by increase in tolerance due to increased frequency of CD4 + CD25 + FoxP3+ regulatory T-cells. Ageing associated oxidative stress and solid tumour associated hypoxia are also associated with an increase in the number and function of regulatory T-cells. This review covers the aspects of redox regulation of Treg differentiation and functions during development, ageing, immunity and stem cell homeostasis. We also propose redox modulation based therapeutic interventions for prevention and treatment of T-cell associated disorders.

Modulation of Caspase-3 activity using a redox active vitamin K3 analogue, plumbagin, as a novel strategy for radioprotection.

Radiation induced damage to normal cells is a major shortcoming of conventional radiotherapy, which necessitates the development of novel radio-protective drugs. An ideal radio-modulator would protect normal cells while having cytotoxic effects on cancer cells. Plumbagin is a potent anti-tumour agent and has been shown to sensitize tumour cells to radiation-induced damage. In the present study, we have evaluated the radio-protective potential of plumbagin and found that it protected normal lymphocytes against radiation-induced apoptosis, but did not protect cancer cells against radiation. Plumbagin offered radioprotection even when it was added to cells after irradiation. The ability of only thiol based antioxidants to abrogate the radio-protective effects of plumbagin suggested a pivotal role of thiol groups in the radio-protective activity of plumbagin. Further, protein interaction network (PIN) analysis was used to predict the molecular targets of plumbagin. Based on the inputs from plumbagin's PIN and in light of its well-documented ability to modulate thiol groups, we proposed that plumbagin may act via modulation of caspase enzyme which harbours a critical catalytic cysteine. Indeed, plumbagin suppressed radiation-induced increase in homogenous caspase and caspase-3 activity in lymphocytes. Plumbagin also inhibited the activity of recombinant caspase-3 and mass spectrometric analysis revealed that plumbagin covalently interacts with caspase-3. Further, the in vivo radioprotective efficacy of plumbagin (single dose of 2mg/kg body weight) was demonstrated by its ability to rescue mice against radiation (7.5 Gy; Whole Body Irradiation) induced mortality. These results indicate that plumbagin prevents radiation induced apoptosis specifically in normal cells by inhibition of caspase-3 activity.

Mitochondrial targeted curcumin exhibits anticancer effects through disruption of mitochondrial redox and modulation of TrxR2 activity.

Mitocurcumin is a derivative of curcumin, which has been shown to selectively enter mitochondria. Here we describe the anti-tumor efficacy of mitocurcumin in lung cancer cells and its mechanism of action. Mitocurcumin, showed 25-50 fold higher efficacy in killing lung cancer cells as compared to curcumin as demonstrated by clonogenic assay, flow cytometry and high throughput screening assay. Treatment of lung cancer cells with mitocurcumin significantly decreased the frequency of cancer stem cells. Mitocurcumin increased the mitochondrial reactive oxygen species (ROS), decreased the mitochondrial glutathione levels and induced strand breaks in the mitochondrial DNA. As a result, we observed increased BAX to BCL-2 ratio, cytochrome C release into the cytosol, loss of mitochondrial membrane potential and increased caspase-3 activity suggesting that mitocurcumin activates the intrinsic apoptotic pathway. Docking studies using mitocurcumin revealed that it binds to the active site of the mitochondrial thioredoxin reductase (TrxR2) with high affinity. In corroboration with the above finding, mitocurcumin decreased TrxR activity in cell free as well as the cellular system. The anti-cancer activity of mitocurcumin measured in terms of apoptotic cell death and the decrease in cancer stem cell frequency was accentuated by TrxR2 overexpression. This was due to modulation of TrxR2 activity to NADPH oxidase like activity by mitocurcumin, resulting in higher ROS accumulation and cell death. Thus, our findings reveal mitocurcumin as a potent anticancer agent with better efficacy than curcumin. This study also demonstrates the role of TrxR2 and mitochondrial DNA damage in mitocurcumin mediated killing of cancer cells.

Baicalein induces cell death in murine T cell lymphoma via inhibition of thioredoxin system.

We have earlier demonstrated the radioprotective potential of baicalein using murine splenic lymphocytes. Here, we have studied the effect of baicalein on murine T cell lymphoma EL4 cells and investigated the underlying mechanism of action. We observed that baicalein induced a dose dependent cell death in EL4 cells in vitro and significantly reduced the frequency of cancer stem cells. Previously, we have reported that murine and human T cell lymphoma cells have increased oxidative stress tolerance capacity due to active thioredoxin system. Hence, we monitored the effect of baicalein on thioredoxin system in EL4 cells. Docking studies revealed that baicalein could bind to the active site of thioredoxin reductase. Baicalein treatment led to significant reduction in the activity of thioredoxin reductase and nuclear levels of thioredoxin-1 thereby increasing ASK1 levels and caspase-3 activity. Interestingly, CRISPR-Cas9 based knock-out of ASK1 or over-expression of thioredoxin-1 abolished anti-tumor effects of baicalein in EL4 cells. Further, baicalein administration significantly reduced intra-peritoneal tumor burden of EL4 cells in C57BL/6 mice. Thus, our study describes anti-tumor effects of baicalein in EL4 cells via inhibition of thioredoxin system.

Dimethoxycurcumin, a metabolically stable analogue of curcumin enhances the radiosensitivity of cancer cells: Possible involvement of ROS and thioredoxin reductase.

Dimethoxycurcumin (DIMC), a structural analogue of curcumin, has been shown to have more stability, bioavailability, and effectiveness than its parent molecule curcumin. In this paper the radiosensitizing effect of DIMC has been investigated in A549 lung cancer cells. As compared to its parent molecule curcumin, DIMC showed a very potent radiosensitizing effect as seen by clonogenic survival assay. DIMC in combination with radiation significantly increased the apoptosis and mitotic death in A549 cells. This combinatorial treatment also lead to effective elimination of cancer stem cells. Further, there was a significant increase in cellular ROS, decrease in GSH to GSSG ratio and also significant slowdown in DNA repair when DIMC was combined with radiation. In silico docking studies and in vitro studies showed inhibition of thioredoxin reductase enzyme by DIMC. Overexpression of thioredoxin lead to the abrogation of radiosensitizing effect of DIMC underscoring the role of thioredoxin reductase in radiosensitization. Our results clearly demonstrate that DIMC can synergistically enhance the cancer cell killing when combined with radiation by targeting thioredoxin system.

Prediction of glutathionylation sites in proteins using minimal sequence information and their experimental validation.

S-glutathionylation of proteins plays an important role in various biological processes and is known to be protective modification during oxidative stress. Since, experimental detection of S-glutathionylation is labor intensive and time consuming, bioinformatics based approach is a viable alternative. Available methods require relatively longer sequence information, which may prevent prediction if sequence information is incomplete. Here, we present a model to predict glutathionylation sites from pentapeptide sequences. It is based upon differential association of amino acids with glutathionylated and non-glutathionylated cysteines from a database of experimentally verified sequences. This data was used to calculate position dependent F-scores, which measure how a particular amino acid at a particular position may affect the likelihood of glutathionylation event. Glutathionylation-score (G-score), indicating propensity of a sequence to undergo glutathionylation, was calculated using position-dependent F-scores for each amino-acid. Cut-off values were used for prediction. Our model returned an accuracy of 58% with Matthew's correlation-coefficient (MCC) value of 0.165. On an independent dataset, our model outperformed the currently available model, in spite of needing much less sequence information. Pentapeptide motifs having high abundance among glutathionylated proteins were identified. A list of potential glutathionylation hotspot sequences were obtained by assigning G-scores and subsequent Protein-BLAST analysis revealed a total of 254 putative glutathionable proteins, a number of which were already known to be glutathionylated. Our model predicted glutathionylation sites in 93.93% of experimentally verified glutathionylated proteins. Outcome of this study may assist in discovering novel glutathionylation sites and finding candidate proteins for glutathionylation.

Nrf2 facilitates repair of radiation induced DNA damage through homologous recombination repair pathway in a ROS independent manner in cancer cells.

Nrf2 is a redox sensitive transcription factor that is involved in the co-ordinated transcription of genes involved in redox homeostasis. But the role of Nrf2 in DNA repair is not investigated in detail. We have employed A549 and MCF7 cells to study the role of Nrf2 on DNA repair by inhibiting Nrf2 using all-trans retinoic acid (ATRA) or by knock down approach prior to radiation exposure (4 Gy). DNA damage and repair analysis was studied by γH2AX foci formation and comet assay. Results suggested that the inhibition of Nrf2 in A549 or MCF7 cells led to significant slowdown in DNA repair as compared to respective radiation controls. The persistence of residual DNA damage even in the presence of free radical scavenger N-acetyl cysteine, suggested that the influence of Nrf2 on DNA repair was not linked to its antioxidant functions. Further, its influence on non-homologous end joining repair pathway was studied by inhibiting both Nrf2 and DNA-PK together. This led to synergistic reduction of survival fraction, indicating that Nrf2 may not be influencing the NHEJ pathway. To investigate the role of homologous recombination repair (HR) pathway, RAD51 foci formation was monitored. There was a significant reduction in the foci formation in cells treated with ATRA or shRNA against Nrf2 as compared to their respective radiation controls. Further, Nrf2 inhibition led to significant reduction in mRNA levels of RAD51. BLAST analysis was also performed on upstream regions of DNA repair genes to identify antioxidant response element and found that many repair genes that are involved in HR pathway may be regulated by Nrf2. Together, these results suggest the involvement of Nrf2 in DNA repair, a hitherto unknown function of Nrf2, putatively through its influence on HR pathway.