Hyaluronic acid: More than a carrier, having an overpowering extracellular and intracellular impact on cancer.

Hyaluronic acid (HA), also named hyaluronan, is an omnipresent component of the tissue microenvironment. It is extensively used to formulate targeted drug delivery systems for cancer. Although HA itself has pivotal influences in various cancers, its calibers are somewhat neglected when using it as delivering platform to treat cancer. In the last decade, multiple studies revealed roles of HA in cancer cell proliferation, invasion, apoptosis, and dormancy through pathways like mitogen-activated protein kinase-extracellular signal-regulated kinase (MAPK/ERK), P38, and nuclear factor kappa-light chain-enhancer of activated B cells (NFκB). A more fascinating fact is that the distinct molecular weight (MW) of HA exerts disparate effects on the same type of cancer. Its overwhelming use in cancer therapy and other therapeutic products make collective research on the sundry impact of it on various types of cancer, an essential aspect to be considered in all of these domains. Even the development of new therapies against cancer needed meticulous studies on HA because of its divergence of activity based on MW. This review will provide painstaking insight into the extracellular and intracellular bioactivity of HA, its modified forms, and its MW in cancers, which may improve the management of cancer.

Extrinsic hyaluronic acid induction differentially modulates intracellular glutamine metabolism in breast cancer stem cells.

The effect of low and high molecular weight hyaluronic acid on glutamine metabolism in luminal and basal breast cancer and cancer stem cells is being investigated. In luminal cell lines (MCF-7), HA enhances the intracellular utilization of gln in redox metabolism and decreases its use in TCA. On the contrary, in MDAMB-231 cells, HA induces the uptake of gln to be utilized in anaplerosis rather than ROS maintenance. However, in MCF-7 CSCs, HA induces up-regulation of xCT, further, it uses gln-derived glutamate for the exchange of cystine, thus maintaining ROS levels through xCT. MDA-MB-231 CSCs reduce the secretion of glutamate in response to HA and decrease the gln flux towards reductive carboxylation. Conclusively, our study demonstrated that although the uptake of gln is enhanced by HA, it is differentially utilized intracellularly in breast cancer cells. This study could significantly influence the therapeutics involving HA and Gln in breast cancer.

Pyruvate kinase M2 in chronic inflammations: a potpourri of crucial protein-protein interactions.

Chronic inflammation (CI) is a primary contributing factor involved in multiple diseases like cancer, stroke, diabetes, Alzheimer's disease, allergy, asthma, autoimmune diseases, coeliac disease, glomerulonephritis, sepsis, hepatitis, inflammatory bowel disease, reperfusion injury, and transplant rejections. Despite several expansions in our understanding of inflammatory disorders and their mediators, it seems clear that numerous proteins participate in the onset of CI. One crucial protein pyruvate kinase M2 (PKM2) much studied in cancer is also found to be inextricably woven in the onset of several CI's. It has been found that PKM2 plays a significant role in several disorders using a network of proteins that interact in multiple ways. For instance, PKM2 forms a close association with epidermal growth factor receptors (EGFRs) for uncontrolled growth and proliferation of tumor cells. In neurodegeneration, PKM2 interacts with apurinic/apyrimidinic endodeoxyribonuclease 1 (APE1) to onset Alzheimer's disease pathogenesis. The cross-talk of protein tyrosine phosphatase 1B (PTP1B) and PKM2 acts as stepping stones for the commencement of diabetes. Perhaps PKM2 stores the potential to unlock the pathophysiology of several diseases. Here we provide an overview of the notoriously convoluted biology of CI's and PKM2. The cross-talk of PKM2 with several proteins involved in stroke, Alzheimer's, cancer, and other diseases has also been discussed. We believe that considering the importance of PKM2 in inflammation-related diseases, new options for treating various disorders with the development of more selective agents targeting PKM2 may appear.

Discovery of boronic acid-based potent activators of tumor pyruvate kinase M2 and development of gastroretentive nanoformulation for oral dosing.

Several studies have established that cancer cells explicitly over-express the less active isoform of pyruvate kinase M2 (PKM2) is critical for tumorigenesis. The activation of PKM2 towards tetramer formation may increase affinity towards phosphoenolpyruvate (PEP) and avoidance of the Warburg effect. Herein, we describe the design, synthesis, and development of boronic acid-based molecules as activators of PKM2. The designed molecules were inspired by existing anticancer scaffolds and several fragments were assembled in the derivatives. 6a-6d were synthesized using a multi-step synthetic strategy in 55-70% yields, starting from cheap and readily available materials. The compounds were selectively cytotoxic to kill the cancerous cells at 80 nM, while they were non-toxic to the normal cells. The kinetic studies established the compounds as novel activators of PKM2 and (E/Z)-(4-(3-(2-((4-chlorophenyl)amino)-4-(dimethylamino)thiazol-5-yl)-2-(ethoxycarbonyl)-3-oxoprop-1-en-1-yl) phenyl)boronic acid (6c) emerged as the most potent derivative. 6c was further evaluated using various in silico tools to understand the molecular mechanism of tetramer formation. Docking studies revealed that 6c binds to the PKM2 dimer at the dimeric interface. Further to ascertain the binding site and mechanism of action, rigorous MD (molecular dynamics) simulations were undertaken, which led to the conclusion that 6c stabilizes the center of the dimeric interface that possibly promotes tetramer formation. We further planned to make a tablet of the developed molecule for oral delivery, but it was seriously impeded owing to poor aqueous solubility of 6c. To improve aqueous solubility and retain 6c at the lower gastrointestinal tract, thiolated chitosan-based nanoparticles (TCNPs) were prepared and further developed as tablet dosage form to retain anticancer potency in the excised goat colon. Our findings may provide a valuable pharmacological mechanism for understanding metabolic underpinnings that may aid in the clinical development of new anticancer agents targeting PKM2.

Comparative stemness and differentiation of luminal and basal breast cancer stem cell type under glutamine-deprivation.

Glutamine (gln) metabolism has emerged as a cancer therapeutic target in past few years, however, the effect of gln-deprivation of bCSCs remains elusive in breast cancer. In this study, effect of glutamine on stemness and differentiation potential of bCSCs isolated from MCF-7 and MDAMB-231 were studied. We have shown that bCSCs differentiate into CD24+ epithelial population under gln-deprivation and demonstrated increased expression of epithelial markers such as e-cadherin, claudin-1 and decreased expression of mesenchymal protein n-cadherin. MCF-7-bCSCs showed a decrease in EpCAMhigh population whereas MDAMB-231-bCSCs increased CD44high population in response to gln-deprivation. The expression of intracellular stem cell markers such sox-2, oct-4 and nanog showed a drastic decrease in gene expression under gln-deprived MDAMB-231-bCSCs. Finally, localization of ß-catenin in MCF-7 and MDAMB-231 cells showed its accumulation in cytosol or perinuclear space reducing its efficiency to transcribe downstream genes. Conclusively, our study demonstrated that gln-deprivation induces differentiation of bCSCs into epithelial subtypes and also reduces stemness of bCSCs mediated by reduced nuclear localization of ß-catenin. It also suggests that basal and luminal bCSCs respond differentially towards changes in extracellular and intracellular gln. This study could significantly affect the gln targeting regimen of breast cancer therapeutics.

Target specific intracellular quantification of etoposide by quadrupole-time of flight based mass spectrometric method.

Etoposide (ETP), a widely used chemotherapeutic agent has an intracellular target site of action. Unfortunately, the concentration of ETP in plasma does not properly reflect the concentration in its intracellular site of action. As per our knowledge, no reported bioanalytical method is available for intracellular quantification of ETP. In this research, we developed an LC-MS/MS method to quantitate ETP in intracellular compartments of MCF-7 cells. The Abcam nuclear extraction kit was used for extracting the nuclear and cytosolic protein from MCF-7 cells. The method showed excellent linearity in the 20-1000 ng/mL range. The intra and inter-day precision (%CV) including LLOQ were found to be in the range of 2.19-16.96% and 6.71-11.21%, respectively, with an accuracy of 86.87 to 110.37% and 93.03 to 100.50%, respectively. The concentration of ETP in nuclear and cytosolic fraction was successfully quantitated using the developed method. The developed method can be applied to understand the efficacy of different formulations based on the intracellular ETP concentration in vitro. It can be considered as a model method for quantification of other similar categories of drugs in their actual intracellular site of action after required optimization in the methodology.

Hyaluronic acid induction on breast cancer stem cells unfolds subtype specific variations in stemness and epithelial-to-mesenchymal transition.

The reoccurrence of breast cancer is a major concern due to presence of cancer stem cells (CSCs). Considering the key role of hyaluronic acid (HA) in modulating the inflammation and cellular migration in cancer, the response of high molecular weight (HMW) and low molecular weight (LMW) HA towards various subtypes of breast cancer and breast cancer stem cells remain elusive. The aim of this study is to determine the effect of exogenous HMW-HA and LMW-HA on stemness of CSCs and epithelial-to-mesenchymal transition which may help in designing HA based therapeutic strategies. LMW-HA induces EMT in MCF-7 more prominently as compared to MDA-MB-231. However, HMW-HA did not show significant changes in the expression of EMT genes. Surprisingly, both HMW-HA and LMW-HA have shown to decrease the expression of EpCAM in MCF-7 cells and decrease the expression of CD44 in MDAMB-231 cells. HA has maintained the native stem cells phenotype of bCSCs isolated from MCF-7 only. The bCSCs isolated form MDAMB-231 showed a decrease in CD44. Luminal subtype has shown to follow Wnt/ß-catenin whereas in the basal subtype localization of CD44 from surface to cytosol was observed in response to HA. Our study has demonstrated that bCSCs in luminal and basal cells follow differential intracellular signaling mechanisms in response to HA. This study could significantly influence the therapeutics involving HA in breast cancer.

Synthetic lethality: a step forward for personalized medicine in cancer.

Synthetic lethality occurs between two genes when silencing of either gene alone enables viable outcomes but inhibition of both is lethal. Understanding context-dependent functioning of synthetic lethality allows therapeutic targeting in cancer. Furthermore, the paradigm shift toward precision medicine to treat cancer necessitates the establishment of biomarkers to help determine which patient populations might respond to specific drug combinations. Elucidating synthetically lethal gene combinations in cancer could establish clinically relevant drug combinations as well as biomarkers to better treat patients. Here, we have reviewed the recent synthetically lethal gene combinations in preclinical and clinical settings and discuss how this approach could help reveal potential biomarkers.

Advancements in Cancer Stem Cell Isolation and Characterization.

Occurrence of stem cells (CSCs) in cancer is well established in last two decades. These rare cells share several properties including presence of common surface markers, stem cell markers, chemo- and radio- resistance and are highly metastatic in nature; thus, considered as valuable prognostic and therapeutic targets in cancer. However, the studies related to CSCs pave number of issues due to rare cell population and difficulties in their isolation ascribed to common stem cell marker. Various techniques including flow cytometry, laser micro-dissection, fluorescent nanodiamonds and microfluidics are used for the isolation of these rare cells. In this review, we have included the advance strategies adopted for the isolation of CSCs using above mentioned techniques. Furthermore, CSCs are primarily found in the core of the solid tumors and their microenvironment plays an important role in maintenance, self-renewal, division and tumor development. Therefore, in vivo tracking and model development become obligatory for functional studies of CSCs. Fluorescence and bioluminescence tagging has been widely used for transplantation assay and lineage tracking experiments to improve our understanding towards CSCs behaviour in their niche. Techniques such as Magnetic resonance imaging (MRI) and Positron emission tomography (PET) have proved useful for tracking of endogenous CSCs which could be helpful in their identification in clinical settings.