Enhanced extracellular vesicles mediated uttroside B (Utt-B) delivery to Hepatocellular carcinoma cell: Pharmacokinetics based on PBPK modelling.

Our prior investigation has confirmed that the anti-hepatocellular carcinoma activity of the plant saponin, specifically Uttroside B (Utt-B), derived from the leaves of Solanum nigrum Linn. This study concentrated on formulating a novel biocompatible nanocarrier utilizing Extracellular vesicles (EVs) to enhance the delivery of plant saponin into cells. The physicochemical attributes of Extracellular Vesicles/UttrosideB (EVs/Utt-B) were comprehensively characterized through techniques such as Transmission Electron Microscopy (TEM) and Fourier-transform infrared spectroscopy (FTIR). Despite the promising therapeutic potential of this uttroside B, mechanistic know-how about its entry into cells is still in its infancy. Our research sheds light on the extracellular vesicle-mediated mechanism facilitating the entry of the saponin into cells, a phenomenon confirmed through the use of by confocal microscopy. We further analysed drug-releasing kinetics and simulated the Pharmacokinetics by PBPK modelling. The simulated pharmacokinetics revealed the bioavailability of Uttroside-B in oral administration against intravenous administration.

Pharmacological attenuation of melanoma by tryptanthrin pertains to the suppression of MITF-M through MEK/ERK signaling axis.

Melanoma is the most aggressive among all types of skin cancers. The current strategies against melanoma utilize BRAFV600E, as a focal point for targeted therapy. However, therapy resistance developed in melanoma patients against the conventional anti-melanoma drugs hinders the ultimate benefits of targeted therapies. A major mechanism by which melanoma cells attain therapy resistance is via the activation of microphthalmia-associated transcription factor-M (MITF-M), the key transcription factor and oncogene aiding the survival of melanoma cells. We demonstrate that tryptanthrin (Tpn), an indole quinazoline alkaloid, which we isolated and characterized from Wrightia tinctoria, exhibits remarkable anti-tumor activity towards human melanoma through the down-regulation of MITF-M. Microarray analysis of Tpn-treated melanoma cells followed by a STRING protein association network analysis revealed that differential expression of genes in melanoma converges at MITF-M. Furthermore, in vitro and in vivo studies conducted using melanoma cells with differential MITF-M expression status, endogenously or ectopically, demonstrated that the anti-melanoma activity of Tpn is decisively contingent on its efficacy in down-regulating MITF-M expression. Tpn potentiates the degradation of MITF-M via the modulation of MEK1/2-ERK1/2-MITF-M signaling cascades. Murine models demonstrate the efficacy of Tpn in attenuating the migration and metastasis of melanoma cells, while remaining pharmacologically safe. In addition, Tpn suppresses the expression of mutated BRAFV600E and inhibits Casein Kinase 2α, a pro-survival enzyme that regulates ERK1/2 homeostasis in many tumor types, including melanoma. Together, we point to a promising anti-melanoma drug in Tpn, by virtue of its attributes to impede melanoma invasion and metastasis by attenuating MITF-M.

The emerging role of selenium metabolic pathways in cancer: New therapeutic targets for cancer.

Selenium (Se) is incorporated into the body via the selenocysteine (Sec) biosynthesis pathway, which is critical in the synthesis of selenoproteins, such as glutathione peroxidases and thioredoxin reductases. Selenoproteins, which play a key role in several biological processes, including ferroptosis, drug resistance, endoplasmic reticulum stress, and epigenetic processes, are guided by Se uptake. In this review, we critically analyze the molecular mechanisms of Se metabolism and its potential as a therapeutic target for cancer. Sec insertion sequence binding protein 2 (SECISBP2), which is a positive regulator for the expression of selenoproteins, would be a novel prognostic predictor and an alternate target for cancer. We highlight strategies that attempt to develop a novel Se metabolism-based approach to uncover a new metabolic drug target for cancer therapy. Moreover, we expect extensive clinical use of SECISBP2 as a specific biomarker in cancer therapy in the near future. Of note, scientists face additional challenges in conducting successful research, including investigations on anticancer peptides to target SECISBP2 intracellular protein.

Pyridine derivatives as anticancer lead compounds with Fatty Acid Synthase as the target: An in silico-guided in vitro study.

For the past few decades, structure-based drug discovery (SBDD) has become an inevitable technique in the drug development process for screening hit compounds against therapeutic targets. Here, we have successfully used the SBDD approach viz. virtual high-throughput screening to identify potential inhibitors against the Ketoacyl synthase (KS) domain of Fatty acid synthase (FASN). Overexpression of FASN, and subsequent enhancement of de novo lipogenesis is a key survival strategy of cancer cells. Hence, targeting lipid metabolism using FASN inhibitors has been considered as a promising method to induce metabolic stress, thereby posing a survival disadvantage to cancer cells. In the present study, we have successfully identified eight FASN inhibitors from Asinex Elite database by implementing in silico tools. Five of the hit compounds share a common ring structure, which enables characteristic binding interactions with FASN-KS. Among them, in vitro validation showed that SFA 22637550 possesses significant FASN inhibitory activity and antiproliferative effect in human cancer cells of various origins. The maximum sensitivity was exhibited towards HepG2 hepatocellular carcinoma cells (IC50 = 28 µM). The mode of cell death was found to be apoptosis with a significant increase in SubG0 population without affecting any other phases of the cell cycle. The current study puts forward an excellent core structure for the development of potent FASN inhibitors for successfully targeting cancer cell metabolism, thereby causing selective cell death.

Activation of the Innate Immune System by Treponema denticola Periplasmic Flagella through Toll-Like Receptor 2.

Treponema denticola is an indigenous oral spirochete that inhabits the gingival sulcus or periodontal pocket. Increased numbers of oral treponemes within this environment are associated with localized periodontal inflammation, and they are also part of an anaerobic polymicrobial consortium responsible for endodontic infections. Previous studies have indicated that T. denticola stimulates the innate immune system through Toll-like receptor 2 (TLR2); however, the pathogen-associated molecular patterns (PAMPs) responsible for T. denticola activation of the innate immune system are currently not well defined. In this study, we investigated the role played by T. denticola periplasmic flagella (PF), unique motility organelles of spirochetes, in stimulating an innate immune response. Wild-type T. denticola stimulated the production of the cytokines tumor necrosis factor alpha (TNF-α), interleukin-1ß (IL-1ß), IL-6, IL-10, and IL-12 by monocytes from human peripheral blood mononuclear cells, while its isogenic nonmotile mutant lacking PF resulted in significantly diminished cytokine stimulation. In addition, highly purified PF were able to dose dependently stimulate cytokine TNF-α, IL-1ß, IL-6, IL-10, and IL-12 production in human monocytes. Wild-type T. denticola and the purified PF triggered activation of NF-κB through TLR2, as determined using a variety of TLR-transfected human embryonic 293 cell lines, while the PF-deficient mutants lacked the ability to stimulate, and the complemented PF-positive T. denticola strain restored the activation. These findings suggest that T. denticola stimulates the innate immune system in a TLR2-dependent fashion and that PF are a key bacterial component involved in this process.

In silico screening for identification of fatty acid synthase inhibitors and evaluation of their antiproliferative activity using human cancer cell lines.

De novo lipogenesis (DNL) by upregulation of fatty acid synthase (FASN) is an important metabolic alteration of cancer cells. FASN is over-expressed in several cancers and is often associated with a high risk of recurrence and poor prognosis. Differential expression of FASN in cancer cells and their normal counterparts leads to the impression that FASN can be an attractive druggable target in cancer therapy. Present study focuses on identification of inhibitors against FASN ketoacyl synthase (KS) domain from Asinex Biodesign compound database using in silico tools. Virtual screening resulted in the identification of two hit compounds BDD27845077 and BDD27845082 with a common core structure. Molecular Docking studies showed that BDD27845077 and BDD27845082 bind at the substrate entry channel of KS domain with GScore -12.03 kcal/mol and -12.29 kcal/mol respectively. Molecular dynamics (MD) simulation of the protein-ligand complexes shows the binding stability of ligands with FASN-KS. In vitro validation of BDD27845082 demonstrated that the compound possesses antiproliferative activity in a panel of human cancer cell lines including MDA-MB-231 (breast cancer), HCT-116 (colon cancer) and HeLa (cervical cancer) with maximum sensitivity against HCT-116 (IC 50 = 25 µM). The study put forward two lead compounds against FASN with favorable pharmacokinetic profile as indicated by virtual screening tools for the development of cancer chemotherapeutics.

Kanamycin Resistance Cassette for Genetic Manipulation of Treponema denticola.

Treponema denticola has been recognized as an important oral pathogen of the "red complex" bacterial consortium that is associated with the pathogenesis of endodontal and periodontal diseases. However, little is known about the virulence of T. denticola due to its recalcitrant genetic system. The difficulty in genetically manipulating oral spirochetes is partially due to the lack of antibiotic resistance cassettes that are useful for gene complementation following allelic replacement mutagenesis. In this study, a kanamycin resistance cassette was identified and developed for the genetic manipulation of T. denticola ATCC 35405. Compared to the widely used ermF-ermAM cassette, the kanamycin cassette used in the transformation experiments gave rise to additional antibiotic-resistant T. denticola colonies. The kanamycin cassette is effective for allelic replacement mutagenesis as demonstrated by inactivation of two open reading frames of T. denticola, TDE1430 and TDE0911. In addition, the cassette is also functional in trans-chromosomal complementation. This was determined by functional rescue of a periplasmic flagellum (PF)-deficient mutant that had the flgE gene coding for PF hook protein inactivated. The integration of the full-length flgE gene into the genome of the flgE mutant rescued all of the defects associated with the flgE mutant that included the lack of PF filament and spirochetal motility. Taken together, we demonstrate that the kanamycin resistance gene is a suitable cassette for the genetic manipulation of T. denticola that will facilitate the characterization of virulence factors attributed to this important oral pathogen.

Folic acid conjugated cross-linked acrylic polymer (FA-CLAP) hydrogel for site specific delivery of hydrophobic drugs to cancer cells.

BACKGROUND: The hydrogel based system is found to be rarely reported for the delivery of hydrophobic drug due to the incompatibility of hydrophilicity of the polymer network and the hydrophobicity of drug. This problem can be solved by preparing semi-interpenetrating network of cross-linked polymer for tuning the hydrophilicity so as to entrap the hydrophobic drugs. The current study is to develop a folic acid conjugated cross-linked pH sensitive, biocompatible polymeric hydrogel to achieve a site specific drug delivery. For that, we have synthesized a folic acid conjugated PEG cross-linked acrylic polymer (FA-CLAP) hydrogel and investigated its loading and release of curcumin. The formed polymer hydrogel was then conjugated with folic acid for the site specific delivery of curcumin to cancer cells and then further characterized and conducted the cell uptake and cytotoxicity studies on human cervical cancer cell lines (HeLa). RESULTS: In this study, we synthesized folic acid conjugated cross-linked acrylic hydrogel for the delivery of hydrophobic drugs to the cancer site. Poly (ethyleneglycol) (PEG) diacrylate cross-linked acrylic polymer (PAA) was prepared via inverse emulsion polymerization technique and later conjugated it with folic acid (FA-CLAP). Hydrophobic drug curcumin is entrapped into it and investigated the entrapment efficiency. Characterization of synthesized hydogel was done by using Fourier Transform-Infrared spectroscopy (FT-IR), Transmission Electron Microscopy (TEM), Differential Scanning Calorimetry (DSC). Polymerization and folate conjugation was confirmed by FT-IR spectroscopy. The release kinetics of drug from the entrapped form was studied which showed initial burst release followed by sustained release due to swelling and increased cross-linking. In vitro cytotoxicity and cell uptake studies were conducted in human cervical cancer (HeLa) cell lines. CONCLUSIONS: Results showed that curcumin entrapped folate conjugated cross-linked acrylic polymer (FA-CLAP) hydrogel showed higher cellular uptake than the non folate conjugated form. So this can be suggested as a better delivery system for site specific release of hydrophobic cancer drugs.

Phytochemicals as Potential Lead Molecules against Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is the most prevalent form of liver cancer, accounting for 85-90% of liver cancer cases and is a leading cause of cancer-related mortality worldwide. The major risk factors for HCC include hepatitis C and B viral infections, along with chronic liver diseases, such as cirrhosis, fibrosis, and non-alcoholic steatohepatitis associated with metabolic syndrome. Despite the advancements in modern medicine, there is a continuous rise in the annual global incidence rate of HCC, and it is estimated to reach >1 million cases by 2025. Emerging research in phytomedicine and chemotherapy has established the anti-cancer potential of phytochemicals, owing to their diverse biological activities. In this review, we report the major phytochemicals that have been explored in combating hepatocellular carcinoma and possess great potential to be used as an alternative or in conjunction with the existing HCC treatment modalities. An overview of the pre-clinical observations, mechanism of action and molecular targets of some of these phytochemicals is also incorporated.

A Novel Combinatorial Regimen Using Sorafenib and Uttroside B, A US FDA-designated 'Orphan Drug', for the Treatment of Hepatocellular Carcinoma.

INTRODUCTION: Sorafenib (Sor) is the first-line treatment option in clinics for treating advanced unresectable hepatocellular carcinoma (HCC). However, acquired chemoresistance and adverse side effects associated with Sor monotherapy limit its clinical benefits. We have previously reported the exceptional anti-HCC potential of uttroside B (Utt-B), a furostanol saponin isolated in our lab from Solanum nigrum Linn. leaves. The current study has evaluated the supremacy of a combinatorial regimen of Sor and Utt-B over Sor monotherapy. METHODS: MTT assay was used for In vitro cytotoxicity studies. A clonogenic assay was conducted to assess the anti-proliferative effect of the combination. Annexin V/PI staining, confocal microscopy, FACS cell cycle analysis, and Western blotting experiments were performed to validate the pro-apoptotic potential of the combination in HepG2 and Huh7 cell lines. Pharmacological safety evaluation was performed in Swiss albino mice. RESULTS: Our results indicate that Utt-B augments Sor-induced cytotoxicity in HepG2 and Huh7 cells. The combination inhibits the proliferation of liver cancer cells by inducing apoptosis through activation of the caspases 7 and 3, leading to PARP cleavage. Furthermore, the combination does not induce any acute toxicity in vivo, even at a dose five times that of the effective therapeutic dose. CONCLUSION: Our results highlight the potential of Utt-B as an effective chemosensitizer, which can augment the efficacy of Sor against HCC and circumvent Sor-induced toxic side effects. Moreover, this is the first and only report to date on the chemosensitizing potential of Utt-B and the only report that demonstrates the therapeutic efficacy and pharmacological safety of a novel combinatorial regimen involving Utt-B and Sor for combating HCC.

Characteristics of Streptococcus mutans genotypes and dental caries in children.

This longitudinal cohort study evaluated the diversity, commonality, and stability of Streptococcus mutans genotypes associated with dental caries history. Sixty-seven 5- and 6-yr-old children, considered as being at high caries risk, had plaque collected from baseline through 36 months for S. mutans isolation and genotyping using repetitive extragenic palindromic-PCR (4,392 total isolates). Decayed, missing, or filled surfaces (dmfs (primary teeth)/DMFS (secondary teeth)) for each child were recorded at baseline. At baseline, 18 distinct genotypes were found among 911 S. mutans isolates from 67 children (diversity), and 13 genotypes were shared by at least two children (commonality). The number of genotypes per individual was positively associated with the proportion of decayed surfaces (p-ds) at baseline. Twenty-four of the 39 children who were available at follow-up visits maintained a predominant genotype for the follow-up periods (stability) and this was negatively associated with the p-ds. The observed diversity, commonality, and stability of S. mutans genotypes represent a pattern of dental caries epidemiology in this high-caries-risk community, which suggests that fewer decayed surfaces are significantly associated with lower diversity and higher stability of S. mutans genotypes.

A randomized study of sodium hypochlorite versus formocresol pulpotomy in primary molar teeth.

BACKGROUND: Alternatives to vital pulpotomy treatment in primary teeth are being sought because of the high formaldehyde content of traditional formocresol (FC) pulpotomy medicaments. AIM: The aim was to compare the clinical and radiographic success of vital pulpotomy treatment in primary molars using 3% sodium hypochlorite (NaOCl) versus a 1:5 dilution of Buckley's FC. DESIGN: Pulpotomies were performed in primary molars of healthy children between 3 and 10 years old. Sixty-five primary teeth were randomized into two groups that were evaluated for treatment outcomes. Following treatment, the pulp chamber was filled with zinc oxide eugenol (ZnOE) and restored with a stainless steel crown cemented with glass ionomer cement. Clinical and radiographic outcomes were recorded at 6 and 12 months. RESULTS: The control (FC) and experimental (NaOCl) groups demonstrated 100% clinical success at 6 and 12 months. The NaOCl group had 86% (19/22) radiographic success at 6 months and 80% (12/15) at 12 months. The FC group had 84% (21/25) radiographic success at 6 months and 90% (9/10) at 12 months. No significant differences were found in the radiographic outcomes between the two groups at 6 and 12 months (Fisher's exact test; P=0.574 and P=0.468, respectively). CONCLUSION: NaOCl demonstrated clinical and radiographic success comparable to FC.

Curcuminoids as Anticancer Drugs: Pleiotropic Effects, Potential for Metabolic Reprogramming and Prospects for the Future.

The number of published studies on curcuminoids in cancer research, including its lead molecule curcumin and synthetic analogs, has been increasing substantially during the past two decades. Insights on the diversity of inhibitory effects they have produced on a multitude of pathways involved in carcinogenesis and tumor progression have been provided. As this wealth of data was obtained in settings of various experimental and clinical data, this review first aimed at presenting a chronology of discoveries and an update on their complex in vivo effects. Secondly, there are many interesting questions linked to their pleiotropic effects. One of them, a growing research topic, relates to their ability to modulate metabolic reprogramming. This review will also cover the use of curcuminoids as chemosensitizing molecules that can be combined with several anticancer drugs to reverse the phenomenon of multidrug resistance. Finally, current investigations in these three complementary research fields raise several important questions that will be put among the prospects for the future research related to the importance of these molecules in cancer research.

Targeting receptor tyrosine kinase signaling: Avenues in the management of cutaneous squamous cell carcinoma.

Non-melanoma skin cancer (NMSC) is the most frequently diagnosed cancer worldwide. Among the various types of NMSCs, cutaneous squamous cell carcinoma (cSCC) exhibits more aggressive phenotype and is also the second-most prevalent type. Receptor tyrosine kinases (RTK) triggers key signaling events that play critical roles in the development of various cancers including cSCC. Unsurprisingly, for this reason, this family of proteins has become the cynosure of anti-cancer drug discovery pipelines and is also being considered as attractive targets against cSCC. Though inhibition of RTKs in cSCC has yielded favourable results, there is still scope for bettering the therapeutic outcome. In this review, we discuss the relevance of RTK signaling in the progression of cutaneous squamous cell carcinoma, and observations from clinical trials that used RTK inhibitors against cSCC. Backed by results from preclinical studies, including those from our lab, we also give insights into the scope of using some natural products as effective suppressors of RTK signaling and skin carcinogenesis.

Targeting BRF2: insights from in silico screening and molecular dynamic simulations.

The Transcription factor II B (TFIIB)­related factor 2 (BRF2) containing TFIIIB complex recruits RNA polymerase III multi-subunit complex to selective gene promoters that altogether are responsible for synthesizing a variety of small non-coding RNAs, including a special type of selenocysteine tRNA (tRNASec), micro-RNA (miRNA), and other regulatory RNAs. BRF2 has been identified as a potential oncogene that promotes cancer cell survival under oxidative stress through its genetic activation. The structure of the BRF2 protein was modeled using the Robetta server, refined, and validated using the Ramachandran plot. A virtual approach utilizing molecular docking was used to screen a natural compound library to determine potential compounds that can interact with the molecular pin motif of the BRF2 protein using Maestro (Schrodinger). Subsequent molecular dynamics simulation studies of the top four ligands that exhibited low glide scores were performed using GROMACS. The findings derived from the simulations, in conjunction with the exploration of hydrogen bonding patterns, evaluation of the free energy landscape, and thorough analysis of residue decomposition, collectively converged to emphasize the robust interaction characteristics exhibited by Ligand 366 (Deacetyl lanatoside C) and ligand 336 (Neogitogenin)-with the BRF2 protein. These natural compounds may be potential inhibitors of BRF2, which could modulate the regulation of selenoprotein synthesis in cancer cells. Targeting BRF2 using these promising compounds may offer a new therapeutic approach to sensitize cancer cells to ferroptosis and apoptosis.Communicated by Ramaswamy H. Sarma.

The role of AMPK in cancer metabolism and its impact on the immunomodulation of the tumor microenvironment.

Adenosine monophosphate-activated protein kinase (AMPK) is a key metabolic sensor that is pivotal for the maintenance of cellular energy homeostasis. AMPK contributes to diverse metabolic and physiological effects besides its fundamental role in glucose and lipid metabolism. Aberrancy in AMPK signaling is one of the determining factors which lead to the development of chronic diseases such as obesity, inflammation, diabetes, and cancer. The activation of AMPK and its downstream signaling cascades orchestrate dynamic changes in the tumor cellular bioenergetics. It is well documented that AMPK possesses a suppressor role in the context of tumor development and progression by modulating the inflammatory and metabolic pathways. In addition, AMPK plays a central role in potentiating the phenotypic and functional reprogramming of various classes of immune cells which reside in the tumor microenvironment (TME). Furthermore, AMPK-mediated inflammatory responses facilitate the recruitment of certain types of immune cells to the TME, which impedes the development, progression, and metastasis of cancer. Thus, AMPK appears to play an important role in the regulation of anti-tumor immune response by regulating the metabolic plasticity of various immune cells. AMPK effectuates the metabolic modulation of anti-tumor immunity via nutrient regulation in the TME and by virtue of its molecular crosstalk with major immune checkpoints. Several studies including that from our lab emphasize on the role of AMPK in regulating the anticancer effects of several phytochemicals, which are potential anticancer drug candidates. The scope of this review encompasses the significance of the AMPK signaling in cancer metabolism and its influence on the key drivers of immune responses within the TME, with a special emphasis on the potential use of phytochemicals to target AMPK and combat cancer by modulating the tumor metabolism.

Corrigendum: Targeting thymidylate synthase enhances the chemosensitivity of triple-negative breast cancer towards 5-FU-based combinatorial therapy.

[This corrects the article DOI: 10.3389/fonc.2021.656804.].

Evaluation of in-office dental unit waterline testing.

In-office dental unit waterline (DUWL) testing systems are commercially available for monitoring DUWL bacteria. The current study compared Aquasafe, Petrifilm, and Heterotrophic Plate Count Sampler (HPCS) with R2A plating methodology, considered the gold standard for enumerating heterotrophic bacteria in potable water. Samples were collected from 20 dental units. Heterotrophic bacterial counts of ≤500 CFUs/mL were used as the cut-off for assessing in-office testing compared to R2A laboratory plating. Validity was assessed using sensitivity and specificity, along with positive and negative predictive values. Results were also compared using concordance and kappa statistics. All in-office tests demonstrated 100% specificity and positive predictive values, while sensitivity and negative predictive values were low (Petrifilm, 57%/50%; HPCS, 50%/46%; Aquasafe, 21%/35%). Concordance and kappa values for agreement with R2A plating were as follows: Petrifilm 70% (κ = 0.44), HPCS 65% (κ = 0.38), and Aquasafe 45% (κ = 0.14). In-office DUWL testing with Aquasafe, Petrifilm, and HPCS agreed poorly with R2A plating methodology and is not valid or reliable as a means of accurately monitoring bacterial density in DUWL. These in-office test systems should not be used for assessing compliance with the ADA and CDC standard for acceptable heterotrophic bacterial counts in DUWLs (≤500 CFUs/mL).