Cell and Tissue Journal

Abstract Introduction: The anther culture technique is used in vitro for many plant species as an effective tool for obtaining haploid and doubled-haploid lines.
Aims: This study aimed to investigate the influence of different concentrations of iron oxide nanoparticles and benzylaminopurine (BAP) on callus formation, embryogenesis, regeneration, and rooting in bell pepper anther culture
Materials and Methods: The experiment was conducted as a factorial experiment in a completely randomized design under in vitro culture conditions. Flower buds of appropriate size (equal sepal to petal ratio or slightly longer petal) were collected from the greenhouse, and acetocarmine solution was used to determine the growth and development stage of microspores. The results showed that the most suitable stages for embryogenesis induction were the late mononuclear and early binuclear stages. In order to sterilize the flower buds, 70% ethanol for 30 seconds and 5% sodium hypochlorite for 20 minutes were used, and after each stage, they were washed three times with sterile distilled water. Then, the anthers were separated from the flower bud and placed in C medium containing 2 mg/L naphthalene acetic acid (NAA), different concentrations of BAP (0, 0.1, 0.5, and 1 mg/L), and different concentrations of iron oxide nanoparticles (0, 1, 10, and 20 mg/L). After that, the explants cultured in C medium were kept at 35°C in a dark place for 8 days in order to apply heat treatment. Then, they were transferred to 25°C in the light for 4 days. After this period, in order to induce embryogenesis, the explants were transferred from C medium to R medium and were subcultured every three weeks until embryos emerged. For further growth and root development, the embryos were transferred to V medium.
Results: The analysis of variance showed that different concentrations of iron oxide nanoparticles had a significant effect on the percentages of embryogenesis, regeneration, and rooting, but had no significant effect on the percentage of callus formation. The results of the mean comparison showed that among the different concentrations of iron oxide nanoparticles, the 1 mg/L treatment produced the highest embryogenesis percentage (11.11%). Furthermore, the mean comparison results for regeneration indicated that the 1 mg/L treatment resulted in the highest regeneration percentage (16.66%). The results of the interaction effects showed that among the different concentrations of iron oxide nanoparticles and BAP, the highest percentage of embryogenesis was observed in the treatment of 20 mg/L iron oxide nanoparticles and 0 mg/L BAP. Also, the treatment of 20 mg/L iron oxide nanoparticles and 0 mg/L BAP had the highest percentage of regeneration (33.33%). After sufficient growth and root formation, the obtained plants were removed from the glass culture containers and transferred to pots containing sterilized culture medium and watered for adaptation. The tops of the pots were covered with plastic cups, and after three days, the cups were pierced, and the plastic was gradually removed from the plant for further adaptation. Ploidy levels were determined by chromosome counting after staining the root tip cells. The results showed that out of the 23 obtained plants, 21 were diploid and had 2n=2x=24 chromosomes, and 2 were haploid and had n=x=12 chromosomes.
Discussion: Applying different concentrations of iron oxide nanoparticles in medium C had a positive effect on the regeneration of bell pepper plants. Iron is a vital micronutrient for several key cellular processes in plants. In addition, iron is essential for ensuring the structural integrity of proteins. Iron nanoparticles positively affect plant growth in tissue culture by increasing morphological parameters. The beneficial effects of nanoparticles on plants are related to their high solubility and reactivity, which affect how they interact with membranes and other cellular components, due to their large specific surface area. The treatment of 20 mg/L of nanoparticles and 0 mg/L of BAP had the highest regeneration percentage. The culture medium containing nano-iron leads to the plant benefiting from the element iron directly at all different stages of growth and its participation with other nutrients, and therefore the production of many nutrients containing it leads to the transfer of their excess to different parts of the plant. The presence of nano iron in the culture medium has led to a nutritional balance in the plant and, as a result, has improved the plant growth process by increasing the amount of nutrients and elements accumulated in the microsample. Cytokinins act as a nutrient reservoir, which leads to an increase in the percentage of elements in the microsample and an increase in the percentage of sugars in the culture medium, which is important in the structural processes in tissues and affects the accumulation of sugars in it, and is reflected in increased vegetative growth.
Conclusion: In bell pepper anther culture, different concentrations of iron oxide nanoparticles, along with plant growth regulators at different concentrations, showed a great effect on embryogenesis, regeneration, and rooting.

Abstract Aim: Di-2-ethylhexyl phthalate (DEHP) is used as plasticizer to produce flexible polyvinyl chloride (PVC) which is used in food and medical industries. Due to temperature change and contact with biological fluids and other liquids, DEHP leaches out from PVC. Humans get exposed to DEHP in different way including food consumption and medical utilities such as blood bags, blood transfusion tubes, syringes. At 2002, 120000 tons of DEHP has been produced in United States of America and this production was raised up to 230000 tons at 2006. In Iran, only in one of the industries called as Farabi petrochemical industries, 55000 tons of phthalate per year is produced. It has been reported that the DEHP concentration in human blood might reach to 52 to 55 µg/ml when blood is stored in polyvinyl chloride bags for two weeks.  Previously, the effect of different concentration of DEHP on viability, proliferation and differentiation of rat bone marrow mesenchymal stem cells was investigated. In the present study, effect of 100 µM (39 µg/ml) of DEHP on the expression of genes related to osteogenic differentiation of rat bone marrow mesenchymal stem cells was investigated.
Material and methods: In this experimental study, rat bone marrow mesenchymal stem cells were extracted from Wistar rats and after 3^rd passage the cells cultured was performed in osteogenic media in presence of 100 µM of DEHP for 21 days. The viability of the cells was studied using 3-[4, 5-dimethylthiazol-2yl]-2, 5-diphenyl-tetrazolium bromide (MTT) assay. In addition, the osteogenic differentiation of the rat bone marrow mesenchymal stem cells was investigated using quantitative alizarin red test and calcium concentration determination. In addition, alkaline phosphatase enzyme activity as a marker of osteogenic differentiation was measured.  Expression of the osteogenic related genes (osteonectin, SMAD1، BMP2، BMP7 and RUNX2) were studied using reverse transcriptase-PCR. Data was analyzed and the minimum level of significant was considered as p<0.05.
Results: Data analysis revealed, rat bone marrow mesenchymal stem cells viability reduced significantly (p<0.05) following treatment with 100 µM of DEHP when compared to control one. Also, we observed a significant (p<0.05) reduction in matrix production based on significant reduction in alizarin red concentration as well as calcium content and alkaline phosphatase enzyme activity. In addition, a significant (p<0.05) reduction in total protein also was observed in the samples extracted from BMSCs differentiated to osteoblasts in presence of DEHP. Meanwhile, a significant (p<0.05) down regulation of osteogenic related genes was confirmed while no change (p>0.05) was observed in the expression of GAPDH.
Conclusion: based on this study, long term exposure to DEHP caused reduction in matrix production which strongly showed the osteogenic differentiation of rat bone marrow mesenchymal stem cells is affected by low concentration of this environmental pollutant. As it was observed, the concentration used in this study was lower than the concentration of this pollutant in blood bags. Therefore, if a patient gets exposed to the biological or non-biological fluids in a treatment procedure, large amount of DEHP would enter the blood circulation system. As DEHP is used in production of food containers and medical utilities such as blood tubing, blood bags, dialysis tubes in dialysis machines, we strongly suggest a legal restriction to be forced on the industrial which using this chemical as plasticizer.

Abstract Aim: This research, design and fabrication of a composite scaffold for growth, proliferation and differentiation of Cardiac Progenitor Cells (CPCs) has been considered.
Material and method: Polycaprolactone / Gelatin composite scaffolds with a ratio of 70:30 and with the most similarities to the cardiac extracellular matrix was fabricated with aligned nanofibers. Using scanning electron microscopy (SEM), mechanical strength analysisand also contact angel test, the scaffold was investigated due to have the most necessary characteristics to be proportional for cardiac scaffold. Finally, By Real time-PCR test, the expression of the specific genes related to the Cardiac Progenitor Cells differentiation (MYH-6, TTN and CX-43) has been analyzed.
Results: Based on our results from contact angle test and mechanical strength experiments, we concluded that our designed scaffold is suitable for the culture of Cardiac Progenitor Cells on it. The Real time-PCR analysis also showed the good expression of genes associated with contraction.
Conclusion: What makes the cardiomyocytes different from other cells is their contraction related to specific cardiac genes being responsible for beating synchronization. The results of this study showed that aligned Polycaprolactone / Gelatin composite scaffold has the appropriate potential for induction of cardiac progenitor cells differentiation and application in heart tissue engineering.
 

Abstract

Abstract Introduction: Pancreatic cancer is the fourth leading cause of cancer-related deaths worldwide. 5-Fluorouracil is one of the commonly used chemotherapeutic drugs. The plant Artemisia absinthium has attracted attention as a potential herbal anticancer agent. This study investigated the effect of the methanolic extract of this plant on the expression of miR-34a and the apoptotic genes BAX, Caspase-3, and Caspase-9 in AsPC-1 pancreatic cancer cells.
Aims: This study aimed to evaluate the cytotoxic and pro-apoptotic effects of Artemisia absinthium methanolic extract on pancreatic cancer cells. The research specifically investigated the molecular mechanism by analyzing expression changes in the tumor suppressor miR-34a and key apoptotic genes BAX, Caspase-3, and Caspase-9 to elucidate the extract's anti-cancer mode of action.
Materials and Methods: To evaluate the cytotoxic effects of the plant extract on cancer cells, an MTT ((3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide)) assay was performed to determine the viability and survival rate of the cells following treatment with various concentrations of the extract, the chemotherapeutic drug fluorouracil (5-FU), and the combined treatment of the extract and the drug. This assay measures cellular metabolic activity and allows quantification of live and dead cells after exposure to different treatments. Based on the obtained results, the IC₅₀ value for each treatment was calculated, representing the concentration at which 50% of the cells were inhibited or killed. After determining the IC₅₀ value, cells were treated with concentrations equal to, lower than, and higher than the IC₅₀ to further investigate the cytotoxic effects and the mode of cell death induced by the treatments. To distinguish between apoptotic and necrotic cell death, the Annexin V-FITC/PI assay was employed. This assay detects phosphatidylserine externalization on the cell membrane and enables differentiation between live, early apoptotic, late apoptotic, and necrotic cells.

In addition to the morphological and physiological assessments, molecular analyses were conducted to examine the expression levels of key apoptosis-related genes, including Caspase-3, Caspase-9, and BAX, as well as the regulatory microRNA miR-34a. Gene expression analysis was performed using Real-time PCR (qPCR).

Results: The results of the MTT assay demonstrated that the proliferation of AsPC-1 pancreatic cancer cells was inhibited by treatment with the extract of Artemisia absinthium and the chemotherapeutic drug fluorouracil (5-FU) in a concentration-dependent manner. As the concentration of each treatment increased, cell viability significantly decreased, indicating a marked cytotoxic effect of both the plant extract and the drug. Moreover, a possible synergistic effect between the extract and fluorouracil in suppressing cell proliferation was observed. To determine the mode of cell death induced by these treatments, the Annexin V-FITC/PI assay was performed. The results revealed that a considerable proportion of treated cells underwent programmed cell death (apoptosis), while the percentage of necrotic cells remained relatively low. These findings suggest that the observed reduction in cell viability is mainly mediated through the activation of apoptotic pathways rather than necrosis. Furthermore, real-time PCR analysis showed a significant upregulation in the expression of the regulatory microRNA miR-34a and the apoptosis-related genes Caspase-3, Caspase-9, and BAX in the treated groups compared to the control group.
Discussion: The obtained results suggest that Artemisia absinthium extract exerts its cytotoxic effect primarily through the induction of apoptosis rather than necrosis in AsPC-1 pancreatic cancer cells. The observed upregulation of miR-34a, Caspase-3, Caspase-9, and BAX implies activation of intrinsic apoptotic pathways. These findings are consistent with previous studies reporting pro-apoptotic properties of A. absinthium and other Artemisia species. Therefore, the extract may enhance the therapeutic response of pancreatic cancer cells when combined with conventional chemotherapeutic agents such as fluorouracil (5-FU).
Conclusion: In conclusion, Artemisia absinthium extract demonstrated strong antiproliferative and apoptosis-inducing effects on AsPC-1 cancer cells in a dose-dependent manner. Its combination with fluorouracil (5-FU) produced a synergistic cytotoxic effect, significantly enhancing cell death through apoptotic signaling. The molecular findings support the potential of this extract as a complementary therapeutic agent. Further studies are recommended to explore its mechanisms and evaluate its efficacy in in vivo models of pancreatic cancer.

Abstract Introduction: Metal nanoparticles exhibit a broad range of properties and have made substantial contributions to the field of biomedicine, particularly concerning targeted drug delivery systems. These nanoparticles function effectively as carriers for a variety of therapeutic agents, including but not limited to antibodies, nucleic acids, chemotherapeutic agents, and peptides. Metals such as silver, gold, zinc, copper, and cerium display remarkable optical characteristics that augment their functional capabilities. Furthermore, their surfaces can be readily modified via hydrogen bonding, covalent bonding, or electrostatic interactions, facilitating their application with bioactive molecules for specific targeting endeavors. The advancement of numerous nanomedicines aimed at tumor treatment has underscored the potential of cerium oxide nanoparticles (CONPs) as a viable therapeutic agent in oncological therapy. Empirical studies suggest that CONPs possess cytotoxic properties against neoplastic cells, inhibit their invasiveness, and enhance their susceptibility to radiation and chemotherapeutic interventions. Additionally, CONPs exhibit minimal toxicity to healthy tissues and contribute to the reduction of reactive oxygen species (ROS) production. Xanthium strumarium, despite its high toxicity, is acknowledged for its medicinal properties. All constituents of the plant contain toxic compounds in varying concentrations and are utilized in the treatment of various ailments, including malaria, cancer, rheumatism, arthritis, tuberculosis, and respiratory allergies, in addition to their analgesic effects. Its roots, fruits, leaves, and other aerial parts possess significant medicinal value. The technique of green synthesis, which employs natural extracts, is utilized for the production of metal or metal oxide nanoparticles.
Aim:The objective of this research is to examine the impact of cerium oxide nanoparticles synthesized through sol-gel and green synthesis techniques utilizing Xanthium strumarium extract on MDA-MB-231 cancer cells.
Materials and Methods: For the green synthesis approach, Zardineh plants, specifically Xanthium classified under the strumarium category with the ALUH 38785 code, were collected from regions adjacent to the Alborz mountains. The leaves of the plant were separated and ground into a fine powder. The resulting powder was extracted and purified using a reflux method. This extract served as a reducing agent for cerium nitrate metal ions. In the sol-gel method, cerium nitrate and cetyltrimethylammonium bromide were used. Characterization of the nanoparticles was conducted using FTIR, EDAX, SEM, and AFM techniques. Furthermore, the MTT assay was used to assess and compare the cytotoxic effects of cerium oxide nanoparticles synthesized on MDA-MB-231 cancerous cells.
Results: AFM analysis indicated that the size of the green synthesized nanoparticles was 10 and Sol-gel method nanoparticles were 13 nm. SEM and EDAX analysis revealed that the synthesized cerium nanoparticles exhibited a nearly spherical shape, with those produced via the sol-gel method showing uniform accumulation and dispersion. The MTT assay results demonstrated that the cytotoxic effect of cerium oxide nanoparticles synthesized on MDA-MB-231 cancerous cells increased with both time and concentration. Notably, the lethal effect of nanoparticles synthesized through the green method was found to be more pronounced than nanoparticles produced via the sol-gel method.
Conclusion: Cerium oxide nanoparticles (CONPs) exert toxicity in cancerous cells, inhibit invasion, and enhance the sensitivity of cancerous cells to radiotherapy and chemotherapy. The green synthesis method uses biological extracts, such as plant materials, in place of industrial chemical agents to reduce metal ions and generate valuable nanoparticles. This approach offers several advantages over traditional chemical synthesis, including cost-effectiveness, reduced environmental pollution, and improved safety for both the environment and human health.

Abstract Aim: Previous studies have shown that Autophagy (lysosome-dependent self-degradation) is upregulated in white adipose tissue of obese subjects. Autophagy-related proteins i.e ATG5 and ATG7 play an essential role in the early stage of the autophagic process. On the other hand, it was found that exercise training modified the bad regulation and maladaptation of white adipose tissue by many molecular mechanisms. Therefore, a question remains to be elucidated whether exercise training can modulate the upregulation of Autophagy induced by a high-fat diet and Autophagy seen in obese subjects. Thus, the present study aimed to survey the effect of regular exercise training on gene expressions of ATG5 and ATG7 in white adipose tissue of mice fed a high-fat diet.
Material and Methods: Twenty-one C57BL/6 male mice (age of four weeks; Approximate body weight of 12 grams) were purchased from the experimental and comparative studies center of Iran University of Medical Sciences. The mice were randomly assigned to three groups: Control (C, n=7), 2) High-fat diet (HFD, n=7), and  High-fat diet with exercise training (HFD-ET, n=7). The mice of the HFD group were fed a high-fat diet (42% kcal of fat) for 12 weeks. The mice of the HFD-ET group were submitted to continuous running on a treadmill for six weeks along with feeding HFD. After the experiment, mice were sacrificed, and visceral adipose tissue pads (epididymal fat) were surgically collected. The Real-Time–PCR methods were used to measure the mRNA expression of ATG5 and ATG7. Data of research were statically analyzed by One-way Analysis of Variance (ANOVA) followed by Tukey's post hoc test.
Results. Data showed that the mRNA expression of ATG5 and ATG7 were higher over two-fold in the HFD group as compared to the control group (p<0.05). Further, the mRNA expression of these genes was higher in the HFD-ET group compared to the control group (p<0.01). Interestingly, the mRNA expression of ATG7 was 1.5 fold higher in the HFD-ET group compared to the HFD group (p<0.05). However, the mRNA expression of ATG5 was not significantly changed in the HFD-ET group as compared to the HFD group (p>0.05).
Conclusion. These results indicate that long-term feeding high-fat diet causes upregulating of the gene expression of key factors involved in the early stage of the autophagy process (i.e ATG5 and ATG7). Regular exercise training could augment the HFD-induced upregulation of ATG7 gene expression. However, it could not change the HFD-induced upregulation of ATG5 gene expression. Based on the results could be speculated that exercise training accompanied by a high-fat diet may more stimulate the autophagy mechanism in white adipose tissue, probably resulting in a positive adaptation in white adipose tissue development. 

Abstract Aim: The lipid compositions of mammalian plasma membrane of spermatozoa are highly sensitive to oxidative stress due to the presence of high amounts of unsaturated fatty acids. If the production of active oxygen compounds is more than the antioxidant capacity of sperm to neutralize its effects, the sperm will undergo oxidative damage, which is characterized by the reduction of qualitative and viability parameters of spermatozoa. In recent years, research has been done on the antioxidant properties of some plants and their protective role for animal spermatozoa. So, the purpose of this research was to investigate the in vitro effect of Syzygium aromaticum extract on the spermatozoa qualitative parameters and preservation of sperm viability under the semen storage in liquid condition.
Material and Methods: Semen collection was done from 8 Arabi rams weekly for 6 weeks and their semen was immediately mixed and after dilution, divided into 5 parts. The treatments included the addition of different levels of zero (control), 25, 50, 75 and 100 µg/ml of clove bud extract to the semen diluent. At zero, 24, 48, 72 and 96 hours after storage the semen samples at 5°C, the quality parameters of the spermatozoa included the motility, viability, plasma membrane integrity and morphological defect rates were evaluated.
Results: At zero time, spermatozoa quality parameters were not affected by the treatments (P<0.05). In 24 hours, the percentage of motility, viability and plasma membrane integrity of spermatozoa in 100 µg/ml of Syzygium aromaticum extract decreased (P<0.05), and lower levels of extract were ineffective on the mentioned parameters of spermatozoa compared to the control. But, after the storage of samples up to 96 hours, 25 and 50 µg/ml of extract significantly improved the percentage of motility, viability and plasma membrane integrity of spermatozoa compared to the control (P<0.05). There was no significant difference in the percentage of spermatozoa morphological abnormalities among the treatments (P<0.05). 
Conclusion: In general, adding 25 µg/ml of Syzygium aromaticum extract to semen diluent of Arabian ram improved the motility, viability and plasma membrane integrity of spermatozoa as well as maintain their viability during the storage of semen under 5˚C. 

Abstract Aim: Alzheimer's is a neurological disorder characterized by cognitive decline, neuron loss, and eventually dementia. On the other hand, studies have shown that physical activity causes synaptic plasticity, improves cognitive performance, increases memory and learning, reduces anxiety and depression, and protects the brain against neuron-destroying diseases in humans and animals. In addition, sumac has a high antioxidant capacity and can be useful in relieving Alzheimer's disease. According to the studies conducted on the positive effect of exercise on cognitive functions and increasing the antioxidant capacity (including receiving sumac) in improving the process of Alzheimer's disease, the effect of these two factors together on the inflammatory factors of Alzheimer's patients has not been investigated. The purpose of the present study was to investigate the effect of a period of endurance training along with sumac supplementation on inflammatory and apoptotic indices in Alzheimer's male rats.
 Material and Methods: The current research is experimental with a post-test and controlled design with a control group and a placebo. 35 rats (with an average age of 4 to 5 weeks and an average weight of 180 to 200 grams) were randomly divided into control group, Alzheimer's disease, Alzheimer's disease with sumac supplementation, Alzheimer's disease with endurance exercise, and Alzheimer's disease with endurance exercise and sumac supplementation. Alzheimer's induction was done by injecting 8 mg/kg of trimethyl tin chloride along with 200 microliters of normal saline. To feed the sumac (Rhus coriaria L)) to rats, the top branch of the sumac plant was ground. The powder obtained from it was mixed with the food of rats at a ratio of ten percent. Then, the mixture was made into a paste and molded into a plate and dried. Endurance training was done in the form of swimming in a special rat pool with dimensions of 80 x 50 x 50 cm, with a water wave maker and water with a temperature of 30 to 33 degrees. Endurance swimming exercises were performed for 12 weeks and 5 days a week. 48 hours after the end of the training program, the rats were anesthetized. 5 ml of blood sample was taken from the heart and transferred to gel tubes. Then the serum was separated by a centrifuge model 5804 manufactured by Eppendorf and transferred to a microtube and a negative twenty-degree freezer. The levels of IL-18, bax, bcl2 and cas3 were analyzed using ELISA method. Data were analyzed using one-way ANOVA test and Tukey's post hoc test (P<0.05).
Results: We found that induction of Alzheimer's disease increases IL-18, bax, bcl2 and cas3 proteins (p=0.001). After 12 weeks of intervention, the level of IL-18, bax, bcl2 and cas3 proteins in the Alzheimer group + endurance exercise was significantly lower than the Alzheimer group (p=0.001). On the other hand, there was no significant difference between Alzheimer's + endurance training and Alzheimer's + endurance + sumac groups in terms of IL-18, bax, bcl2 and cas3 protein levels (p>0.05).
Conclusion: Epidemiological studies suggest the reduction of inflammation in the prevention and treatment of Alzheimer's. However, clinical evidence does not consider the use of anti-inflammatory drugs to be very successful. Both sumac and exercise are strong antioxidants and anti-inflammatory agents, which probably have double positive physiological effects when they are placed next to each other. Our findings suggest that endurance training improves the level of inflammatory indices in Alzheimer's rat, although adding sumac to the exercise program is not likely to improve apoptotic and inflammatory indices.

Abstract Aim: The purpose of the current study is to investigate the cytotoxic and oxidative effects of Salvia officinalis hydroalcoholic extract on cancer A549 cells.
Material and Methods: The cells were seeded in plates to investigate concentrations of Salvia officinalis extract and MTT measurement was done to determine IC50 concentration and cell viability on days 1, 3, 5, and 7. Moreover, analyses of superoxide dismutase and catalase enzymes involved in oxidative stress pathway, and AO/EB staining for a qualitative investigation of cell lines has been conducted in order to explore the effects of IC50 concentration on apoptosis induction. Also, Giemsa and DAPI stainings were utilized to explore the morphological changes of cell and nucleus..
Results: IC50 concentration of Salvia extract for A549 cells was determined 5 mg/mL. According to the results, Salvia extract reduced the cell viability of A549 cells. Based on the results, Salvia extract had a significantly greater cytotoxic effect compared to the control sample of A549 cell line. Treatment cells indicated some clear and dose-dependent differences at different times. The results of stainings proved the apoptosis induction in the treatment group. Furthermore, regarding the enzyme expression results the activity of superoxide dismutase and catalase enzymes in the cell groups treated by Salvia extract was significantly increased, compared to the control group.
Conclusion: Consistent with the results of this study, in addition to the anti-oxidative activity Salvia officinalis hydroalcoholic extract revealed significant apoptotic effects on lung cancer cells, considering a time and dose-dependent method.
 

Abstract

Abstract Aim: In recent years using of Bone marrow mesenchymal stem cells (BMSC) in regenerative medicine, tissue engineering and gene therapy is highly regarded. Convenient access, ability to expand and MSC differentiation capacity along with the ability of adhesion to plastic surfaces and in-vitro growth and development are considered as the characteristic feature of these cells. Bone marrow mesenchymal stem cells possess the ability to differentiate into mesodermal lineage, among other adult cells, can be used in tissue engineering and are good candidates for transplantation. Lovastatin as a lowering cholesterol agent and reducing inflammation, as well as antioxidant and, in particular, neuroprotective effects can be effective in the treatment of neurogenic diseases. The aim of this study was to evaluate the effect of lovastatin on survival, proliferation and expression of GDNF and oct4 genes of Bone marrow mesenchymal stem cells. Lovastatin is presumed to exert their neuroprotective effects by inducing neurotrophic factor gene expression and cell proliferation. Material and methods: In this experimental study, we used 4-6 week adult Wistar rats. The BMSCs were isolated from rat femurs and tibias and cultured in α-MEM. The cell pellet was resuspended in α-MEM supplemented with 10% fetal bovine serum (FBS), 1% penicillin and streptomycin and cultured in 25-cm2 culture flasks at a density of 2 × 104 cells and incubated at 37°C and 5% CO2.  For lovastatin treatment, we exposed MSCs to 1 μM, 5 μm, 10 μm and 15 μm of lovastatin for 24 h. The survival rate of cells was measured by MTT assay. The growth rate and proliferation of cells at 24 hours after culture were assessed by staining with DAPI. The expression of Oct4 and GDNF factors was also evaluated by RT-PCR.
 Results: MSCs were attached to culture plate and were quickly proliferated. In the culture plate, these cells were usually appeared in three forms: small spherical, fusiform and fibroblast-like and flattened. The results of this study indicate that the proliferation rate at 5, 10 and 15 µM lovastatin showed a significant increase compared to control groups (P<0.5). Gene expression density of gelial derived neurotrophic factors (GDNF) and oct4 genes showed that, there were significant differences between MSCs treatment groups and control group (P<0.5). Cell viability and proliferation rate indicate that experimental groups has a higher proliferation rate than control group. Moreover, results showed an increase in mRNA expression for GDNF and Oct4 compared to the control group (P <0.05).
 Conclusion:Therefore, lovastatin can be used to improve the culture of mesenchymal stem cells, which is used for transplantation and cell therapy. BMSCs may be a useful therapeutic agent for the treatment of neurodegenerative disorders.

Abstract Aim: The sperm membrane is rich in unsaturated fatty acids, which is very sensitive to the damage caused by ROS as a result of lipid peroxidation. Using suitable antioxidants in semen extenders can reduce the level of ROS. Rutin is a naturally occurring antioxidant polyphenolic flavonoid with powerful radical scavenging and lipid peroxidation inhibition properties. However, no studies have investigated the potential impact of rutin supplementation in cooling preservation of ram epididymal sperm. The current research was aimed to compare different concentrations of rutin antioxidant in the cooling process.
Material and methods: In this experimental study, testes of mature rams were collected from a local slaughterhouse and transferred to the laboratory within 1 hours in saline (0.9% NaCl) solution. In the laboratory, the epididymis was separated from the testis and cleaned from any connective tissues and blood vessels. For sperm recovery, the cauda epididymis was trimmed with a scalpel and epididymal sperm was obtained by cutting into small pieces in 5 ml of tris base extender pre-warmed at 37°C and incubated for 10 min to allow sperm swim out. The diluted sperm samples were divided into five equal experimental groups including: 1) tris base extender (Control), 2) extender containing 0.5 mM rutin, 3) extender containing 0.75 mM rutin, 4) extender containing 1 mM rutin, and 5) extender containing 1.25 mM rutin. The sperm samples were cooled gradually from 37°C to 4°C in 2 hours and held at 4°C. Sperm motility, viability, membrane integrity and lipid peroxidation were evaluated after extension (0 h) and at 24 and 48 hours of storage. The motility, viability, membrane integrity and morphology of sperm samples were evaluated immediately after extension (0 h) and at 24 and 48 h of storage at 4°C. The sperm motion kinematics were objectively evaluated by using the computer-assisted sperm analysis (CASA) system. The eosin-nigrosin staining technique was applied to evaluate viability. The hypo-osmotic swelling test (HOST) was performed to assess membrane integrity. Sperm morphology was determined by staining sperm smears with Hancock’s solution. Lipid peroxidation was evaluated by measuring malondialdehyde (MDA) concentration.
Results: The data obtained from this experiment show that after 24 and 48 hours of cooling, the addition of rutin antioxidant at a concentration of 0.75 and 1 mM causes a significant increase in total motility, progressive motility and straight path velocity. The results of the present study show that experimental treatments at 0, 24 and 48 hours after cooling have no effect on VAP, VCL and STR. The results of this experiment show that after 24 and 48 hours of cooling, the addition of rutin antioxidants at concentrations of 0.75 and 1 mM significantly increases the viability and integrity of the membrane. Also, the results show that the experimental treatments reduce the amount of MDA. The results show that the applied treatments have no effect on sperm morphology.
Conclusion: The results show that using rutin antioxidant in concentrations of 0.75 and 1 mM improves the quality of ram sperm.

Abstract Aim: The aim of this experimental study was to investigate the effective infertility factors of physoaline compounds on the adult reproductive system of Balb/C adult female.
Material and Methods: The above experiment was performed by preparing the aqueous extract of the plant and selecting three doses of 7.5g/kg,9g/kg and 15g/kg;Simultaneously with the experimental groups,the distilled water sham group was injected and the control group was kept intact.Vaginal stromal smear cycles were prepared to examine different stages.One hour after the last injection,the mice were given blood samples and then killed by chloroform and their uterus and ovaries were prepared for histological studies.
Results: Statistical studies have shown that injecting 7.5g/kg of extract reduces estrogen and restores 33% of fertility in mice. Also, at a dose of 9 g/kg, the mice were initially infertile, but one month after the last injection, 16.5% of the mice showed a return to fertility and a significant increase in progesterone.
Conclusion: According to the results, it is probably because physalines have a pseudo-steroidal structure. The receptor is occupied by physaline and can cause abnormalities in the growth and development of oocytes, uterine tissue cells and ovaries, and infertility.
 

Abstract Aim: Pheasant is a pleasant bird whose long and magnificent tail distinguishes it from other types of birds. The cerebellum is organized in the form of leaves that are located in a row in the rostrocaudal axis of the brain. Myelin is the twisting of the plasma membrane of the oligodendrocyte and Schwann cells around an axon, which insulates it and increases the speed of nerve current conduction. Due to the importance and fundamental role of the nervous system, many studies have been done on the nervous system of mammals and other birds, but no research has been done on the development of the pheasant's nervous system, therefore, this research is proposed.
Material and methods: In this study, sixty fertilized pheasant eggs were used. Healthy eggs were placed in the automatic incubator and head sampling was done on different days of the embryo from the 7th day and the one-day-old chick. The specimens were immediately fixed in 10% neutral buffered formalin solution for 24–48 hours and then submitted to the dehydration process by passing them through a series of ascending ethanol alcohol each for two hours (70, 80, 90 and 100%) and then specimens were cleared in xylene for one hour after that embedded in paraffin wax and then the blocks were sectioned by microtome at 5μm thickness. First, all tissue sections were stained with Hematoxylin and Eosin for histological structure, and then to identify myelin fibers and glial cells, sections were stained with special stain; Luxal Fast Blue Cresyl Etch Violet and Mallory's Phosphotungstic Acid-Hematoxylin dyes.
Results: In the cerebellum of a 7-day-old pheasant fetus, myelin fibers were seen in the form of very thin short strands among the primary nerve cells in an irregular and scattered manner. With increasing fetal age, the density, and probably the length of myelin fibers and the size of nerve cells increased also, the spatial arrangement of myelin fibers was seen in the form of regular thickness and almost from the 17-day embryo of the pheasant onwards, the regular arrangement of these fibers led to the recognition of the white and gray matter of the cerebellum.
In 19-day-old pheasant embryo onwards, the regular arrangement of these fibers caused a clear recognition of the white and gray matter of the cerebellum, which until now from this age, they were not distinguishable, but empty spaces between myelin fibers were observed.
Conclusion: The results showed that myelin and glial cells start to form in the pheasant cerebellum from the age of 7 and as the embryo ages, these cells become bigger and completely fill the space between the neurons. Also, myelin fibers were first observed in the form of very thin and scattered threads at the age of 7 embryos and As the embryo ages, these fibers become denser  and finally, in the white matter of the cerebellum of a day-old chick, they find a spatial arrangement in the form of very thick and regular bundles.

Abstract Introduction: The construction of synthetic pathways within the framework of metabolic engineering is considered a modern approach in biotechnology, enabling the production of valuable compounds from natural biological resources. This strategy focuses on utilizing abundant biomaterials—particularly carbohydrates—for the industrial production of chemical compounds by modifying metabolic pathways in microorganisms. These processes can convert biomass derived from biological sources into fuels, chemicals, and polymers, thereby opening new opportunities for the sustainable production of chemical substances from renewable resources.
Aim: This study specifically focuses on the enzymatic production of benzoylformate decarboxylase (BFD) with the overarching goal of completing the enzymatic pathway for the biosynthesis of BT. This intricate pathway initiates with xylose as the primary carbon source and proceeds through a cascade of four distinct enzymatic reactions. Notably, Escherichia coli (E. coli), possessing two endogenous enzymes integral to this pathway, holds the potential for complete BT biosynthesis upon the introduction of the remaining two requisite genes. This research thus seeks to engineer E. coli as a robust biocatalyst for sustainable BT production. The strategic implementation of a fully functional enzymatic pathway within a well-characterized microbial host, such as  E. coli, promises a more environmentally benign and potentially more efficient route to BT synthesis compared to traditional chemical methods. Furthermore, the ability to manipulate and optimize the expression of these key enzymatic components within E. coli offers opportunities to enhance the overall yield and productivity of the bioproduction process. The successful establishment of such a system could pave the way for large-scale, cost-effective, and sustainable production of this valuable chemical intermediate.
Materials and Methods: To construct an E. coli strain capable of expressing the benzoylformate decarboxylase enzyme, the mdlC gene originating from Pseudomonas putida was amplified and subsequently cloned into both pBAD and pET28 expression vectors. Following the confirmation of successful cloning through rigorous confirmatory assays, protein expression was evaluated using Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE), and the enzymatic activity was assessed.
Results: Benzoylformate decarboxylase (BFD) is a pivotal enzyme within the engineered metabolic pathway for producing 1,2,4-butanetriol (BT) in   E.coli. In this study, the mdlC gene, encoding BFD from Pseudomonas putida, was successfully amplified and cloned into the versatile pBAD and the robust pET28 expression vectors. The pET28  system was preferred due to its ease of use and established track record in protein production, while the pBAD vector was strategically employed for its inducible expression capabilities, allowing for controlled protein synthesis. The expression of the  56 kDa target protein was confirmed through SDS-PAGE analysis, and the enzymatic function in the production of BT was subsequently verified using the sensitive and accurate HPLC method. This work lays a crucial foundation for the further optimization and development of a fully functional and efficient microbial cell factory for the sustainable production of this valuable chemical
Conclusion: The successful transfer of the expression construct into an appropriate E. coli host strain was confirmed by the presence of a distinct protein band at approximately 56 kDa on the SDS-PAGE gel, unequivocally verifying the expression of the mdlC gene. To evaluate the functional capacity of the expressed enzyme, the recombinant vector pBAD.mdlC was transformed into the E. coli TOP10 strain. The subsequent production of BT in the culture medium was meticulously analyzed using High-Performance Liquid Chromatography (HPLC).
                                                           

Abstract

Abstract Aim: The main purpose of the recent study was to evaluate gill chloride cells in Cyprinus carpio during adaptation to different environmental salinities over a short period.
Material and Methods: For this research, Cyprinus carpio by an average weight of 41.4±1 g and length of 16.9±1 cm in four groups with three replications was used. The first group as control was located in fresh water and the next three groups respectively were kept in 4 ppt, 8 ppt, and 12 ppt salinity with the exact condition. On 0, 6, 12, 24, 48, 72, and 96 h the second-gill arch with a maximum thickness of 0.5 cm from the left side was prepared and placed in formalin’s buffer 10%, glutaraldehyde 2.5% solution, and pH 7.4. Then the standard method of preparing tissue sections was performed and paraffin sections with a thickness of 4-6 microns were prepared and stained with hematoxylin and eosin methods and examined with a light microscope. Also, changes in the structure and number of mitochondrial-rich cells, distribution, and dispersion of Na+/K+-ATPase enzyme were studied by scanning electron microscope and immunohistochemical technique.
Results: Results showed obvious modifications in the number and distribution of chloride cells in two filament and lamellar positions at different sampling times. The highest number of chloride cells in the filament and lamella, 26.28±1.33; 19.11±1.27 was reported in 12ppt and the lowest number belonged to the control group. The largest size of chloride cells in the filament and lamella, 22.51±1.56; 12.72±1.32 was reported in 12ppt and the lowest size belonged to the control group.
Conclusion: The findings of this study showed that C. carpio gill chloride cells in the face of different salinities in a short period can change the number and size and adapt to the new condition.

Abstract Aim: The three-dimensional (3D) culture of cancer cells is a method that provides the possibility for growth and comprehensive communication of cells in a 3D space, leading to the generation of tumorspheres. In recent years, developing tumor models from cancer cells by using the 3D cell culture method has attracted a lot of attention because it has been introduced as an accurate and reliable strategy for studying cancer stem cells (CSCs) and CSC-based therapeutics. The 3D tumor models in comparison to the monolayer culture (two-dimensional (2D) culture) of cells more resemble in vivo conditions. Because in tumor models, the tumor microenvironment, cell to cell and cell to extracellular matrix interactions and hypoxia condition, which is necessary for the survival of CSCs, are well reproduced. Through the use of several types of cells, including cancer and stromal cells, tumor models have the ability to develop and reflect the complexity of the tissue of interest, which in such a case, are even more accurate models in reflecting the biochemical and physical conditions of the body. Therefore, in the present study, the 3D model of breast cancer has been constructed with the aim of investigating the relationship between the cell behavior and the cell culture conditions (2D and 3D), and a comparative study of the growth and drug response of the two human breast cancer cell lines; MCF-7, and MDA-MB-231. Material and Methods: The two breast cancer cell lines, MCF-7, and MDA-MB-231, were cultured in 2D and 3D (in two modes; on top and embedded) on the Matrigel-based scaffold. The molecular phenotype of cells based on surface markers was examined by flow cytometry. Mammosphere growth was followed in 12 days and their growth kinetics was determined. To evaluate the drug response of cells, two anticancer drugs; actinomycin D and paclitaxel were applied. Primarily, the IC50 values of the two drugs were evaluated, then the generated mammospheres were treated at the indicated dose of the drugs, and their effect on the growth of the mammospheres was followed.
Results: The MCF-7 and MDA-MB-231 cell lines cultured in 2 and 3D, showed a significant difference in their molecular phenotypes. So, it seems that the expression of CD44 has significantly decreased.  On the other hand, the growth rate of cells in two different modes of 3D culture; on to and embedded, is different. Likely, the drug response evaluation shows a significant difference in 2D and 3D culture, so that the inhibitory effect of paclitaxel compared to actinomycin D has decreased in 3D culture. In addition, the results show that MCF-7 and MDA-MB231 have different drug responses, which can be affected by their different molecular phenotypes. Conclusion: The results of the study confirm that the molecular phenotype of cancer cells, their growth, and drug response are strongly affected by the type of the understudied cell lines, the cell culture method, and the applied drug. Consequently, conducting cancer studies as accurately as possible requires obtaining a model that is most similar to the corresponding tumor in the body.
 

Abstract

Abstract Aim: The current study aims to utilize varicocele induction in male rat models to evaluate effect and impact of varicocele on DNA integrity.
 
Material and Methods: 66 male Wistar rats were divided into 3 groups: control, sham and varicocele-induced (VI). 2 months post-VI, Sperm parameters, percentage of sperm DNA damage, persistent histone and lipid peroxidation were assessed.
 
Results: Animal weight and their epididymal length shown no significant changes but there were significant differences in testis volume and sperm parameters between VI group versus control and sham groups. Similarly, increased level of DNA damage, persistent histone and lipid peroxidation were evaluated, and compared between varicocele and control group.
 
Conclusion: Based on literature and the result of this study, it is likely that increase oxidative stress following varicocele induction leads to lipide peroxidation, DNA damages and reduced sperm parameters. In the current study, by taking the advantage of homogenous genetic background and high number of animals, we showed that varicocele have damaging effect on DNA and chromatin integrity and should be reversed in human before aiming for pregnancy.
 
 

Abstract Aim: About 80% of deaths caused by diabetes are related to cardiovascular diseases. Diabetes and morphine are the main risk factors for cardiovascular diseases that lead to endothelial dysfunction and defective angiogenesis. Today, many angiogenic and angiostatic factors have been identified that play an important role in cardiovascular health, and the most important of them are VEGF (angiogenic factor) and endostatin (angiostatic factor). In this study, the effect of eight weeks of endurance training on vascular endothelial growth factor  (VEGF) and endostatin  (ES) protein levels in the heart tissue of diabetic male Wistar rats with morphine withdrawal syndrome was investigated.
Material and methods: In this experimental study, 32 male Wistar rats were randomly divided into 4 groups of 8 including diabetes control  (D), morphine diabetes  (DM), diabetes + endurance training  (D.ET) and morphine diabetes+ endurance training  (D.M.ET). Then induction of diabetes and addiction to morphine was done. 72 hours after the injection, in order to ensure diabetes, the samples whose blood sugar level was more than 250 mg/dL were considered as diabetic. In order to ensure the dependence of the rats on morphine, at the end of the 21st day, by intraperitoneal injection of naloxone to the samples at the rate of 2 mg/kg of body weight, the signs of withdrawal from addiction such as: jumping, climbing, scratching, teeth grinding , redness around the eyes, diarrhea, tremors, eyelid drooping, erection and standing on two legs for 30 minutes were evaluated. The training groups performed an 8-week endurance training program. At the end of the study, all rats were sacrificed and their heart tissue was removed. VEGF and ES protein levels were measured by ELISA method. Data were analyzed using one-way ANOVA test at a significance level of p≤0.05.
Results: The results showed that in diabetic and morphine rats, the level of VEGF and ES is significantly lower and higher than other groups, respectively  (P<0.05). Also, endurance training is associated with improvement of VEGF and ES values in diabetes + endurance training and morphine diabetes+ endurance training groups  (P<0.05). On the other hand, the level of VEGF and ES in group D with D.M.ET showed no significant difference (P>0.05).
Conclusion: In the present study, it was shown that eight weeks of endurance training caused a significant increase in VEGF in the heart tissue of diabetic rats with morphine withdrawal syndrome. Also, the level of ES protein as one of the most important internal inhibitory factors of angiogenesis decreased significantly. Our findings show that the intervention of endurance training is likely to be effective and useful in regulating the levels of angiogenic factors in the heart tissue of diabetic and diabetic rats in withdrawal syndrome and as a low-cost non-pharmacological solution in the treatment of cardiovascular diseases caused by addiction and diabetes are recommended to specialists in these fields.

Abstract Aim: Potato (Solanum tuberosum L.) is a key crop within the Solanaceae family and ranks as the most significant non-cereal crop globally following major staples such as wheat, rice, and corn. Potatoes can reproduce sexually and asexually via tubers, and plant tissue culture is emerging as an effective method for vegetative propagation, addressing the increasing global demand for agricultural products. Ethylene, a critical plant growth regulator, influences various physiological processes including growth and development. During in vitro culture and due to the wounding of explants, ethylene accumulation can lead to abnormal biological responses, with potato seedlings being susceptible. Thus, investigating the effects of ethylene biosynthesis inhibitors such as pyrazinamide (PZA) and AgNO₃ on potato growth in vitro is essential.
Material and Methods: In this study, potato seedlings were cultivated in Murashige and Skoog (MS) medium, with concentrations of PZA ranging from 0 to 6 mg L⁻¹ and AgNO₃ at 2 mg L⁻¹. After four weeks, the seedlings were harvested and stored at -70°C for later analysis. The growth parameters measured included fresh weight (FW), dry weight (DW), stem and root lengths, leaf area, and leaf and root number. In addition, biochemical parameters, such as photosynthetic pigment levels, total phenol content (TPC), total reactive oxygen species (ROS), and proline concentration were analyzed. Statistical evaluations were conducted using SPSS and PAST software
Results: The results showed that the 2 mg L⁻¹ PZA treatment led to the highest FW and DW and increased leaf numbers; however, it was also correlated with a lower number of rooted plants. Conversely, treatments with 6 mg L⁻¹ PZA promoted longer stem growth, whereas control plants exhibited the largest leaf area, and AgNO₃-treated plants produced the longest roots. The accumulation of H₂O₂ in plants treated with ethylene inhibitors was like controls, but total ROS levels soared by 36% in those treated with 6 mg L⁻¹ PZA compared to controls. This suggests a link between reduced ethylene production, oxidative stress mitigation, and enhanced potato growth. Additionally, total ROS was positively correlated with stem length, but negatively correlated with root length.
Plants use several strategies to combat the damaging effects of ROS, such as the production of antioxidant compounds such as phenolics. Although PZA did not significantly alter TPC compared to controls, treatment with AgNO₃ caused a 61% reduction in TPC. Therefore, PZA did not appear to significantly affect phenolics production in the potato seedlings.
Proline, another critical antioxidant in plants, was found to accumulate significantly in the leaves of plants treated with 6 mg L⁻¹ PZA, which was more than 2.3 times higher than that in controls. This accumulation correlated positively with ROS levels at higher PZA concentrations but showed an inverse relationship with photosynthetic pigment levels.
The PCA revealed the relationships between the measured parameters and the applied elicitors. The samples were categorized into four distinct groups:

Control group: This group primarily exhibited higher FW, DW, and longer roots compared to the treated plants.
Low PZA dose group: These plants displayed elevated levels of photosynthetic pigments, TPC, and leaf area.
Medium PZA dose group: Correlations were observed with an increased number of roots.
6 mg L⁻¹ PZA and AgNO₃ group: These samples contained elevated levels of total ROS and proline.

Conclusion: The study concludes that low concentrations of PZA can stimulate growth while inhibiting ethylene production, resulting in fewer growth abnormalities compared to control plants. However, at elevated PZA concentrations, increased ROS levels may lead to oxidative stress, emphasizing the delicate balance in ethylene's role in plant growth and the necessity for further research to optimize conditions for potato cultivation in vitro. The findings contribute to a deeper understanding of how ethylene inhibitors can enhance potato propagation and possibly other crops in controlled agricultural environments.

Abstract Introduction: Breast cancer is the second leading cause of cancer-related mortality in women. Breast cancer is a multi-step process involving various types of cells, and its prevention remains a global challenge. One of the best ways to prevent breast cancer is through early detection. The upregulation of calcium channels is associated with the proliferation and progression of cancer cells, including breast cancer. The calcium channel blockers are a chemically heterogeneous group that prevents the entry of calcium into the muscle cells of blood vessels and the heart. It has been demonstrated that calcium channel blockers exhibit cytotoxic effects on various types of cancer. Doxorubicin is a well-established chemotherapeutic agent used in the treatment of cancer. Two commonly used calcium channel blockers are amlodipine and diltiazem. However, their interactions with common chemotherapeutic agents such as doxorubicin, which face limitations of cardiotoxicity and cellular resistance, have not been fully investigated.
Aims: This study aimed to evaluate the combined effects of two calcium channel blockers, including amlodipine and diltiazem, on doxorubicin cytotoxicity in breast cancer cell lines.
Materials and Methods: Different concentrations of amlodipine and diltiazem were prepared. Their effects were evaluated both alone and in combination with low concentrations of doxorubicin (which showed minimal cytotoxic effects) on cell proliferation and survival of MDA-MB-231 and MCF-7 human breast cancer cell lines at 48 and 72 hours using the MTT assay. The MTT assay is a colorimetric test that assesses cell metabolic activity. It measures the reduction of MTT, a yellow tetrazole, to purple formazan by mitochondrial enzymes in viable cells. The amount of formazan produced is directly proportional to the number of living cells, making it a useful method for evaluating cell viability and proliferation. Apoptosis is a programmed cell death process that plays a critical role in maintaining tissue homeostasis and eliminating damaged or unwanted cells. Caspase-3/7 activity in drug-treated cell lysates was evaluated to assess apoptosis. All tests were performed at least three times. Differences between samples were analyzed using the t-test and one-way ANOVA, and curves were plotted using Microsoft Excel.
Results: The results of this study demonstrated that both amlodipine and diltiazem inhibited the proliferation of MDA-MB-231 and MCF-7 cancer cells in a concentration- and time-dependent manner. Evaluation of caspase-3/7 activity in the cells treated with amlodipine revealed an increase in caspase-3/7 activity in both cell lines, indicating the induction of apoptosis. In cells treated with diltiazem, caspase-3/7 activity was observed only in MCF-7 cells, while the activity of caspase-3/7 in MDA-MB-231 cells was lower than the control group. Investigating the cytotoxic effect of doxorubicin in the presence of amlodipine and diltiazem showed that these drugs interfered with most cytotoxic concentrations of doxorubicin.
Discussion: The results of this study showed that increasing the concentration of amlodipine and diltiazem, as well as extending the treatment duration, caused cell death in both MDA-MB-231 and MCF-7 cell lines. Caspase-3 and -7 act as executioner enzymes in apoptosis (programmed cell death). The increased activity of caspase 3/7 in MDA-MB-231 and MCF-7 cells treated with amlodipine suggests that amlodipine can induce apoptosis through caspase-dependent pathways. Similarly, increased caspase 3/7 activity was observed in MCF-7 cells treated with diltiazem. However, in MDA-MB-231 cells treated with diltiazem, caspase activity was significantly lower compared to the control group. This unexpected result may indicate that diltiazem induces cell death independently of caspase 3/7 in this cell line, but further experiments are needed to definitively confirm this hypothesis. Calcium is an important regulator of many essential cellular functions and generally acts as a mitogen to stimulate growth in most proliferating cells. It has been reported that tumors typically have abnormally high calcium levels, due to excessive influx of extracellular calcium or the ability of cancerous mitochondria to maintain higher calcium concentrations.  The high levels of intracellular calcium production may activate the calcium second messenger cascade, promoting the overgrowth of certain malignant cells. Human breast cancer cell lines HT-39 and MCF-7, the human promyelocytic leukemia cell line HL-60, and the leukemia cell line L1210, have shown calcium-dependent proliferation. Okazaki et al.  confirmed these findings and demonstrated that the HL-60 cells grow in a manner dependent on extracellular calcium. Additionally, Yonda et al. showed that the growth of a breast cancer cell line (VX2) is tightly regulated by extracellular calcium levels. However, other studies have that removing calcium from the growth medium of some tumorigenic cell lines, such as transformed fibroblasts, hepatic hematomas, mouse embryonic 3T3 cells, and human ovarian cells, does not affect their growth. Therefore, the role of calcium in cell death and proliferation is complicated. Doxorubicin is a widely used chemotherapy drug for breast cancer. Given the high prevalence of hypertension worldwide, cancer patients undergoing chemotherapy often use calcium channel blockers like amlodipine and diltiazem to control blood pressure. Given that amlodipine and diltiazem can induce cancer cell death by blocking calcium channels, this study investigated the cytotoxic effect of doxorubicin in the presence of low concentrations of these drugs on both cell lines. The results showed that amlodipine significantly affected the cytotoxic effect of doxorubicin. This effect depends on concentration and treatment duration. At lower concentrations, amlodipine reduced doxorubicin-induced cell death, and at higher concentrations, due to increased doxorubicin levels, it could not inhibit the drug's toxic effects. Diltiazem is another calcium channel blocker used to lower blood pressure. It has been reported that diltiazem is less potent in lowering blood pressure than amlodipine. Diltiazem also had similar effects to amlodipine. It had antagonistic effects on doxorubicin cytotoxicity at different concentrations and depending on the duration of treatment. Diltiazem, a calcium channel blocker, is known as a P-gp (P-glycoprotein) inhibitor, which reduces cardiotoxicity caused by chemotherapeutic agents. Further studies are needed to explore the underlying mechanisms and therapeutic implications of these findings.
Conclusion: Amlodipine and diltiazem not only induce cell death in cancer cells but also interfere with the cytotoxic effects of doxorubicin at low concentrations. These results highlight the importance of investigating drug interactions between calcium channel blockers and chemotherapeutic agents.

Abstract