Cell and Tissue Journal

Abstract Introduction: Concerns about fossil energy costs, environmental deterioration, and energy security has created strong motivation for the research and development of routes to provide sustainable and renewable fuels. In recent years, the use of biomass to produce highly valued chemicals has attracted widespread attention. Lignocellulosic biomass, as a promising renewable resource for biofuel production, has distinct advantages in terms of economic and environmental benefits. The conversion of renewable raw materials to hydrocarbon fuels is an attractive alternative to fossil fuels from economic and environmental perspectives. The production process of lignocellulosic biomass mainly consists of biomass accumulation, biomass decomposition, simple sugars, and conversion of sugars to biofuel. One of the crucial steps for the economic success of lignocellulosic biofuels depends on the inhibition of competitive metabolism in microorganisms to achieve high productivity. To date, there has been a growing focus on the use of S. cerevisiae and E. coli as cell lines. These two cellular factories have well known advantages. They are genetically transmissible and several tools are available for genetic manipulation. In order to produce xylonate, the engineered xylose is first converted by a dehydrogenase into the intermediate xylonolactone, which is then slowly converted to xylonate in a nonenzymatic reaction.
Aim: The organic compound D-1,2,4-Butanetriol (BT) is a valuable chemical with wide-ranging applications in various fields such as pharmaceuticals, paper, polymer materials, and military applications. However, the chemical synthesis routes for BT have many drawbacks. By genetically modifying microorganisms, the metabolic pathway for producing many substances, including BT, can be engineered. When D-xylose is supplied to the bacterium, it is first converted into an intermediate compound called xylonolactone. This compound slowly converts into xylonate through a non-enzymatic reaction. To produce xylonate, the engineered bacteria receive xylose, which is initially converted by a dehydrogenase reaction catalysed by the xylose dehydrogenase enzyme into an intermediate compound, xylonolactone. Xylonolactone is slowly converted to xylonate in a nonenzymatic reaction. Xylonate is a five-carbon organic acid. Over the past few years, xylonate has increasingly been considered as an important chemical due to its potential as an important chemical component. Xylonate has many applications in the food, chemical, and pharmaceutical industries. Specifically, xylonate can act as a precursor for the synthesis of D-1,2,4-Butanetriol and as a concrete water reducing agent. E. coli was chosen as the target strain for genetic and metabolic engineering due to its fast growth in inexpensive culture media, the presence of two enzymes for BT synthesis, and product formation in less than 24 hours of fermentation. This study aimed to clone and express xylose dehydrogenase from Caulobacter vibrioides in E.coli.
Materials and Methods: At first, to access the bacterial gene sequence, the genome of the target bacterium was extracted. Then, to create a strain expressing the enzymes xylose dehydrogenase and xylonolactonase, the genes for these proteins were amplified from Caulobacter vibrioides CB1 and transferred into E. coli. For this purpose, the target genes were amplified using specifically designed primers via the Polymerase Chain Reaction (PCR) method and initially cloned into a pTZ57cloning vector and then subcloned into pET 26b expression vector. At the final step, the expression of the enzyme was assessed by SDS-PAGE, and the other confirmation was the reduction of NAD+ to NADH, which was used as an activity indicator of the enzyme, as investigated by a change in NADH absorbance at 340 nm.
Results: Confirmatory tests were performed to ensure the presence of the gene in the vectors (using restriction enzymes and colony PCR for gene amplification). The expression and activity of the enzyme were analyzed. The recombinant protein's presence was confirmed by SDS-PAGE for the xylose dehydrogenase gene, with a molecular weight of 52.2 kDa. The estimated expression level of the recombinant protein was approximately 25%.
Conclusion: The objective of this research was solely to establish the metabolic pathway for xylonate production in E. coli by surface expression of enzymes in this pathway (xylose dehydrogenase). The results obtained in this study confirm that half of the pathway is active at the cell surface, but further experiments are required to determine the precise production levels and complete the pathway.

Abstract Introduction: Prostate cancer is one of the most prevalent malignancies among men worldwide and represents a major cause of cancer-related morbidity and mortality. Despite advances in screening and therapeutic approaches, resistance to standard treatments, including androgen‑deprivation therapy, remains a major clinical challenge in prostate cancer. As a result, identifying novel compounds that can suppress proliferation or modulate key molecular regulators of the cell cycle is of considerable interest. Papaverine is an isoquinoline alkaloid derived from the opium poppy, widely known for its long‑standing clinical use as a smooth‑muscle relaxant and vasodilator. In recent years, it has gained attention for its potential anticancer effects, including its influence on mitochondrial function, cellular energy metabolism, and the modulation of signaling pathways involved in cell cycle regulation. However, the specific effects of papaverine on prostate cancer cells have not yet been clearly elucidated. Tamoxifen, a selective estrogen receptor modulator widely used in breast cancer therapy, has also demonstrated off-target antiproliferative effects in various tumor models.
Aims: The present study aimed to evaluate the effects of papaverine and tamoxifen on the viability and molecular regulatory profile of the DU145 prostate cancer cell line. Specifically, the study investigated the cytotoxic potential of these compounds at different concentrations and incubation times, and their ability to alter the expression of the CDK4 gene, a key mediator of G1-to-S phase progression, along with two microRNAs, miR-146a and miR-22, known to influence cell cycle control and tumorigenic pathways. By integrating cellular and molecular findings, the study sought to clarify whether these compounds could serve as potential modulators of prostate cancer cell growth.
Materials and Methods: The DU145 human prostate cancer cell line was cultured under standard conditions and treated with different concentrations of papaverine and tamoxifen. Cell viability was assessed for 24, 48, and 72 hours using the MTT (3- (4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide) assay. Dose–response curves were generated to calculate IC50 values for each compound at the specified time points. To investigate molecular changes, total RNA was extracted from treated and control cells, and the expression levels of the CDK4 gene, miR-146a, and miR-22 were quantified by Real-Time PCR with appropriate internal controls. Relative expression changes were determined using the 2^-ΔΔCt method. The data were analyzed using SPSS software. Results were presented as Mean ± SD, and a significance level of p ≤ 0.05 was considered statistically significant.
Results: Treatment of the DU145 prostate cancer cell line with various concentrations of papaverine and tamoxifen resulted in a significant reduction in cell viability in a concentration- and time-dependent manner, as measured by the MTT assay for 24, 48, and 72 hours. IC50 values were successfully determined for both compounds across the different time points.
Molecular analysis using Real-Time PCR showed that treatment with either papaverine or tamoxifen led to a marked downregulation of CDK4 expression compared with untreated controls. In contrast, both compounds induced a significant up‑regulation of miR‑146a and miR‑22 levels.
Discussion: The observed decrease in viability suggests that papaverine and tamoxifen exert notable antiproliferative effects on DU145 prostate cancer cells. The downregulation of the CDK4 gene provides a mechanistic link to cell cycle arrest, as CDK4 is a central regulator of G1‑to‑S phase progression. Such suppression is in line with reduced cellular proliferation and supports the therapeutic potential of these compounds.
The increased expression of miR‑146a and miR‑22 further highlights the involvement of tumor suppressive microRNAs in mediating these effects. Both miRNAs have been implicated in regulating pathways associated with proliferation, apoptosis, and oncogenic signaling. Their upregulation may contribute to the inhibition of cell growth through negative modulation of critical oncogenic targets.
These results align with prior evidence demonstrating tamoxifen’s antiproliferative effects, but also importantly emphasize that the molecular and cellular impacts of papaverine in prostate cancer remain poorly understood. The current findings provide initial insight into papaverine’s interaction with cell cycle regulatory genes and microRNAs, suggesting a potential role in prostate cancer growth inhibition. Considering the adverse effect profile associated with tamoxifen, papaverine may therefore represent a more favorable candidate for further investigation, particularly as a potential alternative with a potentially safer therapeutic window.
Conclusion: In conclusion, both papaverine and tamoxifen effectively reduced DU145 cell viability and modulated the expression of the CDK4 gene, miR‑146a, and miR‑22, indicating their capability to interfere with cell cycle regulatory mechanisms. While their antiproliferative effects were broadly comparable, the established side effects of tamoxifen suggest limitations in its clinical applicability. Given that papaverine produced similar molecular outcomes without the same known adverse profile, it may be considered a potentially more suitable candidate for further preclinical evaluation in prostate cancer research.