Assessment of human embryonic stem cells differentiation into definitive endoderm lineage on the soft substrates.

Pluripotent stem cells (PSCs) hold enormous potential for treating multiple diseases owing to their ability to self-renew and differentiate into any cell type. Albeit possessing such promising potential, controlling their differentiation into a desired cell type continues to be a challenge. Recent studies suggest that PSCs respond to different substrate stiffness and, therefore, can differentiate towards some lineages via Hippo pathway. Human PSCs can also differentiate and self-organize into functional cells, such as organoids. Traditionally, human PSCs are differentiated on stiff plastic or glass plates towards definitive endoderm and then into functional pancreatic progenitor cells in the presence of soluble growth factors. Thus, whether stiffness plays any role in differentiation towards definitive endoderm from human pluripotent stem cells (hPSCs) remains unclear. Our study found that the directed differentiation of human embryonic stem cells towards endodermal lineage on the varying stiffness did not differ from the differentiation on stiff plastic dishes. We also observed no statistical difference between the expression of yes-associated protein (YAP) and phosphorylated YAP. Furthermore, we demonstrate that lysophosphatidic acid, a YAP activator, enhanced definitive endoderm formation, whereas verteporfin, a YAP inhibitor, did not have the significant effect on the differentiation. In summary, our results suggest that human embryonic stem cells may not differentiate in response to changes in stiffness, and that such cues may not have as significant impact on the level of YAP. Our findings indicate that more research is needed to understand the direct relationship between biophysical forces and hPSCs differentiation.

The symbiotic effect of osteoinductive extracellular vesicles and mineralized microenvironment on osteogenesis.

The increasing prevalence of bone-related diseases has raised concern about the need for an osteoinductive and mechanically stronger scaffold-based bone tissue engineering (BTE) alternative. A mineralized microenvironment, similar to the native bone microenvironment, is required in the scaffold to recruit and differentiate local mesenchymal stem cells at the bone defect site. Further, extracellular vesicles (EVs), pre-osteoblasts' secretome, contain osteoinductive cargo and have recently been exploited in bone regeneration. This work developed a cell-free and mechanically strong interpenetrating network-based scaffold for BTE by combining the action of osteoinductive EVs with a mineralized microenvironment. The MC3T3 (a pre-osteoblast cell line) is used as a source of EVs and as the target population. The optimal concentration of MC3T3-EVs was first determined to induce osteogenesis in target cells. The osteoinductive potential of the scaffold was estimated in vitro by osteogenesis-related markers like the alkaline phosphatase (ALP) enzyme and calcium content. The MC3T3-EVs cargo was also studied for osteoinductive signals such as ALP, calcium, and mRNA. The findings of this work indicated that MC3T3-EVs at a 90 µg/mL dose had significantly higher ALP activity than 0 µg/mL (1.47-fold), 10 µg/mL (1.41-fold), and 30 µg/mL (1.39-fold) EV-concentration on day 14. Further combination of the optimum dose of EVs with a mineralized microenvironment significantly enhanced ALP activity (1.5-fold) and mineralization (3.36-fold) as compared to the control group on day 7. EV cargo analysis revealed the presence of calcium, the ALP enzyme, and the mRNAs necessary for osteogenesis and angiogenesis. ALP activity was significantly boosted in the EV-containing target cells as early as day 1, and mineralization began on day 7 because MC3T3-EVs carry ALP enzymes and calcium as cargo. When osteoinductive EVs were combined with an osteoconductive mineralized microenvironment, osteogenesis was significantly enhanced in target cells at early time points. The interaction between osteoinductive EVs and the mineralized milieu facilitates the process of osteogenesis in the target cells and suggests a potential cell-free strategy for in vivo bone repair.

Retinoic acid signaling is critical for generation of pancreatic progenitors from human embryonic stem cells.

Retinoic acid (RA) is essential for gut endoderm development and has been extensively used for in vitro pancreatic differentiation from human pluripotent stem cells. However, the gene regulatory network triggered by RA signaling remains poorly addressed. Also, whether RA signals control histone modifiers such as the Polycomb group proteins during pancreatic specification remains to be explored. Here, we assess the role of RA on pancreas-specific genes during the differentiation of human embryonic stem cells (hESCs). We demonstrate that RA helps cells exit the definitive endoderm stage and proceed toward a pancreatic fate. Inhibition of the RA pathway using the pharmacological inhibitor LE135 impairs the induction of pancreatic endoderm (PE) markers FOXA2, HNF4α, HNF1ß, HHEX, and PDX1. We further determine that RA signals alter the expression of epigenetic-associated genes BMI1 and RING1B in the hESC-derived pancreatic progenitors. These findings broaden our understanding of the mechanisms that drive early PE specification.

The Molecular Mechanisms Involved in the Hypertrophic Scars Post-Burn Injury.

Scar formation is a normal response to skin injuries. During the scar-remodeling phase, scar tissue is usually replaced with normal, functional tissue. However, after deep burn injuries, the scar tissue may persist and lead to contractures around joints, a condition known as hypertrophic scar tissue. Unfortunately, current treatment options for hypertrophic scars, such as surgery and pressure garments, often fail to prevent their reappearance. One of the primary challenges in treating hypertrophic scars is a lack of knowledge about the molecular mechanisms underlying their formation. In this review, we critically analyze studies that have attempted to uncover the molecular mechanisms behind hypertrophic scar formation after severe burn injuries, as well as clinical trials conducted to treat post-burn hypertrophic scars. We found that most clinical trials used pressure garments, laser treatments, steroids, and proliferative inhibitors for hypertrophic scars, with outcomes measured using subjective scar scales. However, fundamental research using human burn injury biopsies has shown that pathways such as Transforming Growth factor ß (TGFß), Phosphatase and tensin homolog (PTEN), and Toll-like receptors (TLRs) could be potentially regulated to reduce scarring. Therefore, we conclude that more testing is necessary to determine the efficacy of these molecular targets in reducing hypertrophic scarring. Specifically, double-blinded clinical trials are needed, where the outcomes can be measured with more robust quantitative molecular parameters.

Soft substrate maintains stemness and pluripotent stem cell-like phenotype of human embryonic stem cells under defined culture conditions.

Human embryonic stem cells (hESCs) are derived from the inner cell mass (ICM) of the pre-implantation blastocyst. Prior to embryo implantation, the ICM cells are surrounded by trophoblasts which have mechanical stiffness ranging from Pascal (Pa) to kilopascal (kPa). However, under in vitro conditions these cells are cultured on stiff tissue culture treated plastic plates (TCP) which have stiffness of approximately 1 gigapascal (GPa). This obvious dichotomy motivated us to investigate the fate of hESCs cultured on softer substrate, and to probe if the hESCs undergo differentiation or they retain pluripotency on soft substrates. We investigated the expression of pluripotency markers, and lineage-specific markers; we particularly looked at the expression of transcriptional coactivator YAP (Yes-associated protein), an important mediator of extracellular matrix (ECM) mechanical cues and a known downstream transducer of Hippo pathway. Downregulation of YAP has been correlated to the loss of multipotency of human mesenchymal stem cells (hMSCs) and pluripotency in mouse ESCs (mESCs); but we report that hESCs maintain their stemness on soft substrate of varying stiffness. Our findings revealed that on soft substrate hESCs express pluripotency markers and does not undergo substrate-mediated differentiation. Interestingly we show that hESCs maintained basal level of YAP expression for cell survival and proliferation, but YAP expression does not correlate directly with pluripotency in hESCs. To summarize, our results show that hESCs retain their stemness on soft substrate despite downregulation of YAP. Supplementary Information: The online version contains supplementary material available at 10.1007/s10616-022-00537-z.

ChIP-qPCR for Polycomb Group Proteins During Neuronal Differentiation of Human Pluripotent Stem Cells.

Neuronal differentiation is an intricate and a complex process which involves crosstalk among various signaling pathways, growth factors, transcription factors, and epigenetic modifiers. During different stages of neuronal development, there are various histone modifiers which drive the expression of lineage-specific genes. Polycomb group proteins are one of the histone modifiers that control transcriptional repression of specific genes in development, differentiation, and functionality of various tissues. Chromatin immunoprecipitation (ChIP) is a widely used technique to investigate the interaction of proteins and DNA; ChIP combined with quantitative real-time PCR (qPCR) gives a quantitative data about the occupancy of specific protein on a particular stretch of DNA, and this can help us investigate how a protein regulates expression of a specific gene. In this chapter, we describe a protocol for ChIP coupled to qPCR during early neuronal differentiation to identify the specific genomic targets regulated by components of Polycomb repressive complex 1.

Differential expression of retinoic acid alpha and beta receptors in neuronal progenitors generated from human embryonic stem cells in response to TTNPB (a retinoic acid mimetic).

Retinoic acid (RA), an active metabolite of vitamin A, plays a critical role in the morphogenesis and differentiation of various tissues, especially in the central nervous system. RA is the most commonly used morphogen for the differentiation of human embryonic stem cells (hESCs) into neuronal progenitor cells (NPCs), an abundant source of healthy neuronal tissues for regenerative therapy. During the differentiation process, the activity of RA is governed by the involvement of RA receptor subtypes (RAR α, ß, and γ) and their isoforms in the nucleus. However, little is known about the involvement of specific RAR subtypes during neuronal differentiation in humans. It is essential to elucidate the dynamic function of different RAR subtypes and their influence on the phenotypic outcome. Here in this study, we used TTNPB, an analog and stabilized form of retinoic acid that potently and selectively activates retinoic acid receptors. Here we determined the optimum concentration of TTNPBfor the efficient generation of early NPCs from hESCs. Using the optimized concentration of -TTNPB, we found that RARα is the functionally dominant subtype and controls the RA-mediated neurogenesis of hESCs. Importantly, we also found that the RARγ subtype also played a role in neuronal differentiation. In contrast, the RARß subtype negatively correlates with neuronal differentiation. Therefore, pharmacological inhibition of RARß in the TTNPB-mediated differentiation process could be used as a strategy to generate a large number of NPCs in vitro. In summary, our results show that RARα and RARγ play a vital role in the TTNPB-mediated neuronal differentiation of hESCs, -whereas RARß acts as a negative regulator.

Deterministic role of sonic hedgehog signalling pathway in specification of hemogenic versus endocardiogenic endothelium from differentiated human embryonic stem cells.

Embryonic stem cells (ESCs) have been shown to have an ability to form a large number of functional endothelial cells in vitro, but generating organ-specific endothelial cells remains a challenge. Sonic hedgehog (SHH) pathway is one of the crucial developmental pathways that control differentiation of many embryonic cell types such as neuroectodermal, primitive gut tube and developing limb buds; SHH pathway is important for functioning of adult cell of skin, bone, liver as well as it regulates haematopoiesis. Misregulation of SHH pathway leads to cancers such as hepatic, pancreatic, basal cell carcinoma, medulloblastoma, etc. However, its role in differentiation of human ESCs into endothelial cells has not been completely elucidated. Here, we examined the role of SHH signalling pathway in endothelial differentiation of hESCs by growing them in the presence of an SHH agonist (purmorphamine) and an SHH antagonist (SANT-1) for a period of 6 days. Interestingly, we found that activation of SHH pathway led to a higher expression of set of transcription factors such as BRACHYURY, GATA2 and RUNX1, thus favouring hemogenic endothelium; whereas inhibition of SHH pathway led to a reduced expression of set of markers such as RUNX1 and BRACHURY, and an increased expression of set of markers - NFATC1, c-KIT, GATA4, CD31 & CD34, thus favouring endocardiogenic endothelium. The results of this study have revealed the previously unreported deterministic role of SHH pathway in specification of endothelial cells differentiated from human ESCs into hemogenic vs. endocardiogenic lineage; this finding could have major implications for clinical applications.

Placenta: A gold mine for translational research and regenerative medicine.

Stem cell therapy has gained much impetus in regenerative medicine due to some of the encouraging results obtained in the laboratory as well as in translational/clinical studies. Although stem cells are of various types and their therapeutic potential has been documented in several studies, mesenchymal stromal/stem cells (MSCs) have an edge, as in addition to being multipotent, these cells are easy to obtain and expand, pose fewer ethical issues, and possess immense regenerative potential when used in a scientifically correct manner. Currently, MSCs are being sourced from various tissues such as bone marrow, cord, cord blood, adipose tissue, dental tissue, etc., and, quite often, the choice depends on the availability of the source. One such rich source of tissue suitable for obtaining good quality MSCs in large numbers is the placenta obtained in a full-term delivery leading to a healthy child's birth. Several studies have demonstrated the regenerative potential of human placenta-derived MSCs (hPMSC), and most show that these MSCs possess comparable, in some instances, even better, therapeutic potential as that shown by human bone marrow-derived (hBMSC) or human umbilical cord-derived (hUC-MSC) MSCs. The placenta can be easily sourced from the OB/GYN department of any hospital, and if its derivatives such as hPMSC or their EVs are produced under GMP conditions, it could serve as a gold mine for translational/clinical research. Here, we have reviewed recent studies revealing the therapeutic potential of hPMSC and their extracellular vesicles (EVs) published over the past three years.

Sonic hedgehog signals hinder the transcriptional network necessary for pancreatic endoderm formation from human embryonic stem cells.

Hedgehog morphogens govern multiple aspects of pancreas organogenesis and functioning with diverse outcomes across species. Although most current differentiation protocols repress Sonic hedgehog (SHH) signals during in vitro endocrine specification, the role and mechanisms through which the SHH pathway antagonizes pancreas development during in vitro human embryonic stem (hES) cell differentiation remain unclear. We modulated SHH signaling at transitory stages of hES cell-derived pancreatic progenitors and analyzed the effect on cellular fate decisions. We identify the Hedgehog pathway as a negative regulator of pancreatic endoderm formation through up-regulation of a set of pancreatobiliary markers required for ductal specification, including SOX17, FOXA2, HNF1ß, HNF6, PDX1, and SOX9. Surprisingly, active Hedgehog signals impeded a group of pancreatic epithelium markers, including HNF4α, HHEX, PAX6, and PTF1α. To understand how SHH signals repress the transcription of these specific markers, we analyzed Polycomb group proteins. We found differential expression of Polycomb Repressive Complex 1 subunit, BMI1 upon Shh pathway modulation in the pancreatic progenitors. Ectopic activation of Sonic hedgehog results in over-expression of BMI1 and its associated repressive histone mark, H2AK119Ub1, in the multipotent progenitors. Our data suggest that Sonic hedgehog restricts the pancreatic differentiation program by limiting progenitor cells acquiring pancreatic epithelial fates and instead promotes pancreatobiliary differentiation. We further provide mechanistic cues of an association between Hedgehog signaling and epigenetic silencers during pancreatic lineage decisions.

Effect of Sonic hedgehog pathway inhibition on PDX1 expression during pancreatic differentiation of human embryonic stem cells.

Differentiation processes for generating pancreatic progenitors from pluripotent stem cells inhibit Sonic hedgehog signaling through synthetic antagonists. However, the effect of sonic hedgehog inhibition in differentiating human embryonic stem cells remains unclear. The primary aim of this study was to understand the effect of Sonic hedgehog inhibition on key pancreas-specific transcription factors during differentiation of human embryonic stem cells towards a pancreatic lineage. We differentiated human embryonic stem (ES) cells towards the pancreatic progenitor stage. To analyze the effect of Sonic hedgehog inhibition, we differentiated human ES cells in the presence or absence of pathway antagonist, cyclopamine, using the same concentration (0.25 µM) as reported earlier. Changes in gene expression between the groups were examined by quantitative reverse-transcription PCR and immunoblot analyses. Surprisingly, we found that expression of key transcription factors, PDX1 and SOX9, was not majorly affected by inhibition of Sonic hedgehog signals. Effects of inhibiting Hedgehog signals on pancreas-specific markers in differentiating human embryonic stem cells were analyzed in the study. We identified that the expression of pancreas-specific PDX1 and SOX9 was not affected by the Sonic hedgehog pathway in pancreatic progenitor populations from human ES cells. Thus, the restrictive nature of Hedgehog signaling during the early stages of pancreas formation could be facilitated through a transcriptional network beyond PDX1 and SOX9.

Biomaterials Regulate Mechanosensors YAP/TAZ in Stem Cell Growth and Differentiation.

Tissue-resident stem cells are surrounded by a microenvironment known as 'stem cell niche' which is specific for each stem cell type. This niche comprises of cell-intrinsic and -extrinsic factors like biochemical and biophysical signals, which regulate stem cell characteristics and differentiation. Biochemical signals have been thoroughly studied however, the effect of biophysical signals on stem cell regulation is yet to be completely understood. Biomaterials have aided in addressing this issue since they can provide a defined and tuneable microenvironment resembling in vivo conditions. We review various biomaterials used in many studies which have shown a connection between biomaterial-generated mechanical signals and alteration in stem cell behaviour. Researchers probed to understand the mechanism of mechanotransduction and reported that the signals from the extracellular matrix regulate a transcription factor yes-associated protein (YAP)/transcriptional coactivator with PDZ-binding motif (TAZ), which is a downstream-regulator of the Hippo pathway and it transduces the mechanical signals inside the nucleus. We highlight the role of the YAP/TAZ as mechanotransducers in stem cell self-renewal and differentiation in response to substrate stiffness, also the possibility of mechanobiology as the emerging field of regenerative medicines and three-dimensional tissue printing.

PRC1 catalytic unit RING1B regulates early neural differentiation of human pluripotent stem cells.

BACKGROUND: Polycomb group (PcG) proteins are histone modifiers which control gene expression by assembling into large repressive complexes termed - Polycomb repressive complex (PRC); RING1B, core catalytic subunit of PRC1 that performs H2AK119 monoubiquitination leading to gene repression. The role of PRC1 complex during early neural specification in humans is unclear; we have tried to uncover the role of PRC1 in neuronal differentiation using human pluripotent stem cells as an in vitro model. RESULTS: We differentiated both human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) towards neural progenitor stage evident from the expression of NESTIN, TUJ1, NCAD, and PAX6. When we checked the total expression of RING1B and BMI1, we saw that they were significantly upregulated in differentiated neural progenitors compared to undifferentiated cells. Further, we used Chromatin Immunoprecipitation coupled with qPCR to determine the localization of RING1B, and the repressive histone modification H2AK119ub1 at the promoters of neuronal specific genes. We observed that RING1B localized to and catalyzed H2AK119ub1 modification at promoters of TUJ1, NCAM, and NESTIN during early differentiation and later RING1B was lost from its promoter leading their expression; while functional RING1B persisted significantly on mature neuronal genes such as IRX3, GSX2, SOX1, NEUROD1 and FOXG1 in neural progenitors. CONCLUSION: The results of our study show that PRC1 catalytic component RING1B occupies neuronal gene promoters in human pluripotent stem cells and may prevent their precocious expression. However, when neuronal inductive signals are given, RING1B is not only removed from neuronal gene promoters, but the inhibitory H2AK119ub1 modification is also lost.

Pancreas development and the Polycomb group protein complexes.

The dual nature of pancreatic tissue permits both endocrine and exocrine functions. Enzymatic secretions by the exocrine pancreas help digestive processes while the pancreatic hormones regulate glucose homeostasis and energy metabolism. Pancreas organogenesis is defined by a conserved array of signaling pathways that act on common gut progenitors to bring about the generation of diverse cell types. Multiple cellular processes characterize development of the mature organ. These processes are mediated by signaling pathways that regulate lineage-specific transcription factors and chromatin modifications guiding long-term gene expression programs. The chromatin landscape is altered chiefly by DNA or histone modifications, chromatin remodelers, and non-coding RNAs. Amongst histone modifiers, several studies have identified Polycomb group (PcG) proteins as crucial determinants mediating transcriptional repression of genes involved in developmental processes. Although PcG-mediated chromatin modifications define cellular transitions and influence cell identity of multipotent progenitors, much remains to be understood regarding coordination between extracellular signals and their impact on Polycomb functions during the pancreas lineage progression. In this review, we discuss interactions between sequence-specific DNA binding proteins and chromatin regulators underlying pancreas development and insulin producing ß-cells, with particular focus on Polycomb group proteins. Understanding such basic molecular mechanisms would improve current strategies for stem cell-based differentiation while also help elucidate the pathogenesis of several pancreas-related maladies, including diabetes and pancreatic cancer.

Clocking the circadian genes in human embryonic stem cells.

Multicellular organisms respond to changing environment which is primarily driven by light from the sun. Essential cyclical processes such as digestion, sleep, migration and breeding are controlled by set of genes know as circadian genes. The core circadian genes comprise of CLOCK, BMAL-1, PERIOD and CYRPTOCHROME that are expressed cyclically and they regulate expression of several genes downstream. The expression of circadian genes has been well studied in multicellular animals; however, it has been shown that stem cells also possess active circadian cycle genes. The circadian cycle genes have been studied in mouse embryonic stem cells and in adult human stem cells. However, there are only few reports of circadian cycle genes in human pluripotent stem cells. We used human embryonic stem cells to investigate the expression of CLOCK, BMAL-1, PERIOD and CYRPTOCHORME genes by RT-PCR at 6, 18 and 22 hours in undifferentiated and differentiated cells. We differentiated human embryonic stem cells spontaneously by adding 10% fetal bovine serum (FBS), and the cells primarily differentiated into ectoderm and mesoderm. We report that CLOCK and BMAL-1 are differentially expressed while PERIOD and CRYPTOCHROME show cyclicity in differentiated and undifferentiated cells. Our results show circadian genes are active in human embryonic stem cells and this needs to be further investigated as human pluripotent stem cells have potential to be used for cell therapy, where they need to synchronize with the body's circadian cycle.

Inhibition of RING1B alters lineage specificity in human embryonic stem cells.

Polycomb group (PcG) proteins are histone modifiers which are known to perform transcriptional repression and have been shown to be critical during murine embryonic development. PcGs are broadly characterized into polycomb repressive complex 1 (PRC1) and 2 and (PRC2). RING1B, core catalytic unit of PRC1 performs H2AK119 monoubiquitination leading to transcriptional repression. We used human embryonic stem cell (hESC) line to study the fate of pluripotent stem cells (PSCs) under inhibition of RING1B, as its role in human development is still to be completely explored. Embryoid bodies (EBs) were generated to differentiate hESCs using hanging drop method. PRT4165 (synthetic RING1B catalytic activity inhibitor) was added to undifferentiated and differentiated cells for 24 h. When we inhibited RING1B in undifferentiated cells, OCT4 levels remained unchanged indicating RING1B does not regulate pluripotency. The drug when added to differentiated cells led to decrease in the levels of RING1B, BMI1, and H2AK119ub1. Interestingly, we also report that the differentiated cells show an increased expression of neuroectodermal markers: SOX1 and PAX6 as well as expression of other neuroectodermal markers such as TUJ1, FOXG1, and NCAM. However, there was reduction in expression of endodermal (SOX17 and FOXA2) mesodermal marker BRACHYURY and TBX5 in treated EBs compared with control EBs. In summary, alteration of RING1B catalytic activity in hESCs during differentiation promotes neuroectodermal differentiation thus, we demonstrate critical role of RING1B in regulating neural differentiation. The strategy of inhibiting RING1B could be used to direct PSCs towards early neuronal fate.

Polycomb repressive complex 1: Regulators of neurogenesis from embryonic to adult stage.

Development of vertebrate nervous system is a complex process which involves differential gene expression and disruptions in this process or in the mature brain, may lead to neurological disorders and diseases. Extensive work that spanned several decades using rodent models and recent work on stem cells have helped uncover the intricate process of neuronal differentiation and maturation. There are various morphological changes, genetic and epigenetic modifications which occur during normal mammalian neural development, one of the chromatin modifications that controls vital gene expression are the posttranslational modifications on histone proteins, that controls accessibility of translational machinery. Among the histone modifiers, polycomb group proteins (PcGs), such as Ezh2, Eed and Suz12 form large protein complexes-polycomb repressive complex 2 (PRC2); while Ring1b and Bmi1 proteins form core of PRC1 along with accessory proteins such as Cbx, Hph, Rybp and Pcgfs catalyse histone modifications such as H3K27me3 and H2AK119ub1. PRC1 proteins are known to play critical role in X chromosome inactivation in females but they also repress the expression of key developmental genes and tightly regulate the mammalian neuronal development. In this review we have discussed the signalling pathways, morphogens and nuclear factors that initiate, regulate and maintain cells of the nervous system. Further, we have extensively reviewed the recent literature on the role of Ring1b and Bmi1 in mammalian neuronal development and differentiation; as well as highlighted questions that are still unanswered.

Anticancer activity of methylene blue via inhibition of heat shock protein 70.

INTRODUCTION: Heat shock protein 70 (Hsp70) and heat shock protein 90 (Hsp90) chaperones are indispensable to lung cancer cells for their survival and proliferation. In this study we evaluated and compared anticancer potential of methylene blue (MB) as an Hsp70 inhibitor, novobiocin (NB) a well-known Hsp90 inhibitor and their combination. METHODS: In vitro evaluation was done by cell viability assays, fluorescent staining, and flow cytometry analysis using A549 non-small cell lung cancer cells. In vivo anticancer activity was investigated by evaluating oxidative stress, tumor biomarkers, weight, lung microarchitecture, and Hsp70 and Hsp90 inhibitions via immunoblotting in benzo[a]pyrene induced lung carcinogenesis mice model. RESULTS: Using A549 NSCLC cells, we found MB demonstrated lower cell viability versus NB. Together, MB + NB resulted in further decrease in cell viability. SRB assay revealed significantly superior and similar potency for MB versus NB and MB + NB (1:1) versus MB, respectively. Fluorescent staining and flow cytometry analysis displayed early apoptosis by MB (11.4%); early and late apoptosis by MB + NB (13.8%). In vivo, MB significantly inhibited Hsp70. Furthermore, MB significantly alleviated tumor biomarkers (ADA and LDH) and improved lung histopathological features more than NB. Additionally, MB significantly improved SOD, not more than MB + NB or NB and improved LPO. CONCLUSION: MB demonstrated potent anticancer activity in vitro and in vivo via inhibition of Hsp70 in benzo[a]pyrene induced lung carcinogenesis in mice.