Current Pharmaceutical Biotechnology - Volume 22, Issue 9, 2021

Conventional cancer therapeutic approaches broadly include chemotherapy, radiation therapy and surgery. These established approaches have evolved over several decades of clinical experience. For a complex disease like cancer, satisfactory treatment remains an enigma for the simple fact that the causal factors for cancer are extremely diverse. In order to overcome existing therapeutic limitations, consistent scientific endeavors have evolved several potential therapeutic approaches, majority of which focuses essentially on targeted drug delivery, minimal concomitant ramification, and selective high cytotoxicity. The current review focuses on highlighting some of these potential alternatives that are currently in various stages of in vitro, in vivo, and clinical trials. These include physical, chemical and biological entities that are avidly being explored for therapeutic alternatives. Some of these entities include suicide gene, micro RNA, modulatory peptides, ultrasonic waves, free radicals, nanoparticles, phytochemicals, and gene knockout, and stem cells. Each of these techniques may be exploited exclusively and in combination with conventional therapeutic approaches thereby enhancing the therapeutic efficacy of the treatment. The review intends to briefly discuss the mechanism of action, pros, and cons of potential alternatives to conventional therapeutic approaches.

On a worldwide scale, the outbreak of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has led to extensive damage to the health system as well as the global economy. Hitherto, there has been no approved drug or vaccine for this disease. Therefore, the use of general antiviral drugs is at the first line of treatment, though complicated with limited effectiveness and systemic side effects. Given the pathophysiology of the disease, researchers have proposed various strategies not only to find a more specific therapeutic way but also to reduce the side effects. One strategy to accomplish these goals is to use CRISPR/Cas13 system. Recently, a group of scientists has used the CRISPR/Cas13 system, which is highly effective in eliminating the genome of RNA viruses. Due to the RNA nature of the coronavirus genome, it seems that this system can be effective against the disease. The main challenge regarding the application of this system is to deliver it to the target cells efficiently. To solve this challenge, it seems that using virosomes with protein S on their membrane surface can be helpful. Studies have shown that protein S interacts with its specific receptor in target cells named Angiotensin-Converting Enzyme 2 (ACE2). Here, we propose if CRISPR/Cas13 gene constructs reach the infected cells efficiently using a virosomal delivery system, the virus genome will be cleaved and inactivated. Considering the pathophysiology of the disease, an important step to implement this hypothesis is to embed protein S on the membrane surface of virosomes to facilitate the delivery of gene constructs to the target cells.

Stem Cells (SCs) show a wide range of applications in the treatment of numerous diseases, including neurodegenerative diseases, diabetes, cardiovascular diseases, cancer, etc. SC related research has gained popularity owing to the unique characteristics of self-renewal and differentiation. Artificial Intelligence (AI), an emerging field of computer science and engineering, has shown potential applications in different fields like robotics, agriculture, home automation, healthcare, banking, and transportation since its invention. This review aims to describe the various applications of AI in SC biology, including understanding the behavior of SCs, recognizing individual cell type before undergoing differentiation, characterization of SCs using mathematical models and prediction of mortality risk associated with SC transplantation. This review emphasizes the role of neural networks in SC biology and further elucidates the concepts of machine learning and deep learning and their applications in SC research.

Background: Nowadays, medicines derived from natural sources have drawn much attention as potential therapeutic agents in the suppression and treatment of cancer because of their low toxicity and fewer side effects. Objective: The present review aims to assess the currently available knowledge on the ethnomedicinal uses and pharmacological activities of bioactive compounds obtained from medicinal mushrooms towards cancer treatment. Methods: A literature search has been conducted for the collection of research papers from universally accepted scientific databases. These research papers and published book chapters were scrutinized to retrieve information on ethnomedicinal uses of mushrooms, different factors involved in cancer cell proliferation, clinical and in silico pharmaceutical studies made for possible treatments of cancer using mushroom derived compounds. Overall, 241 articles were retrieved and reviewed from the year 1970 to 2020, out of which 98 relevant articles were finally considered for the preparation of this review. Results: This review presents an update on the natural bioactive substances derived from medicinal mushrooms and their role in inhibiting the factors responsible for cancer cell proliferation. Along with it, the present review also provides information on the ethnomedicinal uses, solvents used for extraction of anti-cancer metabolites, clinical trials, and in silico studies that were undertaken towards anticancer drug development from medicinal mushrooms. Conclusion: The present review provides extensive knowledge on various anti-cancer substances obtained from medicinal mushrooms, their biological actions, and in silico drug designing approaches, which could form a basis for the development of natural anti-cancer therapeutics.

Background: The selection of a suitable signal peptide that can direct recombinant proteins from the cytoplasm to the extracellular space is an important criterion affecting the production of recombinant proteins in Escherichia coli, a widely used host. Nanobodies are currently attracting the attention of scientists as antibody alternatives due to their specific properties and feasibility of production in E. coli. Objective: CD44 nanobodies constitute a potent therapeutic agent that can block CD44/HA interaction in cancer and inflammatory diseases. This molecule may also function as a drug against cancer cells and has been produced previously in E. coli without a signal peptide sequence. The goal of this project was to find a suitable signal peptide to direct CD44 nanobody extracellular secretion in E. coli that will potentially lead to optimization of experimental methods and facilitate downstream steps such as purification. Methods: We analyzed 40 E. coli derived signal peptides retrieved from the Signal Peptide database and selected the best candidate signal peptides according to relevant criteria including signal peptide probability, stability, and physicochemical features, which were evaluated using signalP software version 4.1 and the ProtParam tool, respectively. Results: In this in silico study, suitable candidate signal peptide(s) for CD44 nanobody secretory expression were identified. CSGA, TRBC, YTFQ, NIKA, and DGAL were selected as appropriate signal peptides with acceptable D-scores, and appropriate physicochemical and structural properties. Following further analysis, TRBC was selected as the best signal peptide to direct CD44 nanobody expression to the extracellular space of E. coli. Conclusion: The selected signal peptide, TRBC is the most suitable to promote high-level secretory production of CD44 nanobodies in E. coli and potentially will be useful for scaling up CD44 nanobody production in experimental research as well as in other CD44 nanobody applications. However, experimental work is needed to confirm the data.

Background: In recent years, the incidence of sudden deafness has gradually increased, with a very limited understanding of its etiology and pathogenesis. Glucocorticoids are the first choice of the treatment, but some hormone-resistant patients are not sensitive to glucocorticoid therapy. The pathogenesis is not yet known. In this study, we aim to construct the HEI-OC1 cell line stably overexpressing Glucocorticoid Receptor Beta (GRβ), and identify its exact role in the cases of glucocorticoidresistant sudden deafness. Methods: We used the endotoxin lipopolysaccharide-stimulated cochlear hair cells (HEI-OC1) to investigate the relationship of inflammation factor IL-2, TNF alpha, and SRp30c with the high expression GRβ. We built a stable GRβ high expression HEI-OC1 cell line and clarified its effects on the therapeutic effect of dexamethasone. MTT assay, colony formation assay, CCK-8 assay, Western blot, and RT-qPCR were utilized for characterizations. Results: Dexamethasone reduced the LPS-induced inflammatory response from HEI-OC1 cells (p<0.05), detected by MTT assay. Dexamethasone could protect HEI-OC1 cells, but its protective effect was weakened due to the transfection of SRp30c over-expression plasmid (p<0.05). The transfection of SRp30c over-expression plasmid in HEI-OC1 cells could elevate the expressions of GRβ (p<0.05). Conclusion: We clarified the mechanisms of high expression of GRβ in glucocorticoid-resistant sudden sensorineural hearing loss, and proved that the inhibition of SRp30c may act as a new treatment way of glucocorticoid-resistant sudden sensorineural hearing loss.

Background: The consistently increasing reports of bacterial resistance and the reemergence of bacterial epidemics have inspired the health and scientific community to discover new molecules with antibacterial potential continuously. Frog-skin secretions constitute bioactive compounds essential for finding new biopharmaceuticals. The exact antibacterial characterization of dermaseptin related peptides derived from Agalychnis annae, is limited. The resemblance in their conserved and functionally linked genomes indicates an unprecedented opportunity to obtain novel bioactive compounds. Objective: In this study, we derived a novel peptide sequence and determined its antibacterial potentials. Methods: Consensus sequence strategy was used to design the novel and active antibacterial peptide named 'AGAAN' from skin secretions of Agalychnis annae. The in-vitro activities of the novel peptide against some bacterial strains were investigated. Time kill studies, DNA retardation, cytotoxicity, betagalactosidase, and molecular computational studies were conducted. Results: AGAAN inhibited P. aeruginosa, E. faecalis, and S. typhimurium at 20 μM concentration. E. coli and S. aureus were inhibited at 25 μM, and lastly, B. subtilis at 50 μM. Kinetics of inactivation against exponential and stationary growing bacteria was found to be rapid within 1-5 hours of peptide exposure, depending on time and concentration. The peptide displayed weak hemolytic activity between 0.01%-7.31% at the antibacterial concentrations. AGAAN efficiently induced bacterial membrane damage with subsequent cell lysis. The peptide's DNA binding shows that it also targets intracellular DNA by retarding its movement. Our in-silico molecular docking analysis displayed a strong affinity to the bacterial cytoplasmic membrane. Conclusion: AGAAN exhibits potential antibacterial properties that could be used to combat bacterial resistance.

Background: An affibody molecule obtained from a bioengineered staphylococcal protein was previously shown to act as an affinity binder for a wide range of targets and develop Tumour Necrosis Factor α (TNF-α)-binding clones. Methods: In this study, we demonstrated that affibody molecules against TNF-α could bind to recombinant TNF-α on the membrane for biochemical detection. In addition, we examined whether the affibody molecules could block binding between recombinant TNF-α and its receptor on MH7A synovial cells. Results: When a TNF-α-binding affibody was added, the production level of inflammatory mediators IL-6 and MMP-3 in MH7A were found to decrease up to 44%. Additionally, proliferation of synovial cells was also inhibited by the addition of TNF-α to cultivation media. Conclusion: These results suggest that affibody molecules against TNF-α could be candidate molecules for the detection of TNF-α during biochemical analysis and pharmacotherapy for rheumatoid arthritis.

Background: Cecropin-B (Cec-B) is an Antimicrobial Peptide (AMP) found in insects. Objectives: Recombinant production of Cec-B peptide in Escherichia coli (Rosetta™ DE3), and studying its anticancer effect on Hepatocellular Carcinoma Cell line (HCC). Methods: The Cec-B gene of Drosophila melanogaster was synthesized by PCR assembly using the Simplified Gene Synthesis (SGS) method. To express the recombinant peptide in E. coli (Rosetta™ DE3); the synthesized gene was cloned into pET-15b expression vector. The recombinant peptide was expressed as insoluble aggregates called Inclusion Bodies (IBs) using 2mM lactose inducer. IBs were solubilized in a denatured form using 8 M urea followed by in-vitro protein refolding using rapid dilution method. The refolded Cec-B was purified using cation-exchange SP-FF column. Cytotoxicity of recombinant Cec-B (rCec-B) was reported on normal human lung cell line (WI-38), and Hepatocellular carcinoma cell line (HepG2). Results: The Cec-B gene was expressed and purified at concentration 1.212±0.1 mg/ml which represents 48.49±4% of the total proteins injected to the column (2.5±0.2 mg/ml). The safe dose of purified rCec-B on normal WI-38 cells was calculated to be 1.57 mg/ml. The half-maximal Inhibitory Concentration (IC50) of rCec-B on HepG2 cell line was calculated to be 25 μg/ml. Scanning Electron Microscope (SEM) showed that untreated and treated HepG2 cells had cell diameters from 11-12.92 μm and 14.18-21.58 μm, respectively. Conclusion: The results of this study revealed a successful expression of the rCec-B peptide using a pET-based expression system with a simple purification step. The purified peptide could be considered as a hopeful anticancer drug against HCC.

Background: Hepatocellular Carcinoma (HCC) is a primary liver cancer with high mortality. Paeoniflorin is a pinane monoterpene picroside with anti-tumor effect isolated from Chinese peony root and white peony root. Objective: The study was conducted to investigate the underlying mechanism of Paeoniflorin (PF) regulating Hepatocellular Carcinoma (HCC) progression via 5-hydroxytryptamine receptor 1D (5-HT1D). Methods: HepG2 and SMMC-7721 hepatoma cells were treated with different concentrations of PF (0, 5, 10, 20 μM). Cell proliferation, apoptosis, migration, and invasion were examined by CCK-8 and colony formation assays, flow cytometry, wound healing assay, and transwell assay, respectively. RTqPCR assay was used to detect the expression level of 5-HT1D, and Western blot assay was used to detect the expressions of 5-HT1D and Wnt/β-catenin pathway-related proteins. Results: With the increase in PF concentration, the mRNA levels of 5-HT1D in HepG2 and SMMC- 7721 hepatoma cells were decreased in a dose-dependent manner, and the proliferation, colony formation, migration and invasion ability of cells were gradually weakened, while the apoptosis rate was gradually increased. Overexpression of 5-HT1D significantly promoted the proliferation, colony formation, migration and invasion of HepG2 and SMMC-7721 cells, and increased the expression of Wnt/β-catenin pathway-related proteins, β -actenin, survivin, C-myc, and Cyclin D1. Furthermore, 5-HT1D overexpression could reverse the effect of PF on hepatoma cells and inhibit the expressions of Wnt/β-catenin pathway-related proteins. Conclusion: PF may inhibit the progression of HCC by blocking Wnt/β-catenin pathway expression through downregulating 5-HT1D.

Background: Nanomaterials have recently been identified for their potential benefits in the areas of medicine and pharmaceuticals. Among these nanomaterials, silver nanoparticles (Ag-NPs) have been widely utilized in the fields of diagnostics, antimicrobials, and catalysis. Objective: To investigate the potential utility of Citrobacter freundii in the synthesis of silver Nanoparticles (Ag-NPs), and to determine the antimicrobial activities of the Ag-NPs produced. Methods: Aqueous Ag+ ions were reduced when exposed to C. freundii extract and sunlight, leading to the formation of Ag-NPs. Qualitative microanalysis for the synthesized Ag-NPs was done using UVvis spectrometry, Energy Dispersive X-ray analysis (EDX), and scanning and transmission electron microscopy. The hydrodynamic size and stability of the particles were detected using Dynamic Light Scattering (DLS) analysis. The Ag-NPs’ anti-planktonic and anti-biofilm activities against Staphylococcus aureus and Pseudomonas aeruginosa, which are two important skin and wound pathogens, were investigated. The cytotoxicity on human dermal fibroblast cell line was also determined. Results: Ag-NPs were spherical with a size range between 15 to 30 nm. Furthermore, Ag-NPs displayed potent bactericidal activities against both S. aureus and P. aeruginosa and showed noticeable anti-biofilm activity against S. aureus biofilms. Ag-NPs induced minor cytotoxic effects on human cells as indicated by a reduction in cell viability, a disruption of plasma membrane integrity, and apoptosis induction. Conclusion: Ag-NPs generated in this study might be a future potential alternative to be used as antimicrobial agents in pharmaceutical applications for wound and skin related infections.