دانشگاه اراک سلول و بافت 2228-7035 16 2 2025 06 22 Expression of the Benzoylformate Decarboxylase Enzyme Gene in Escherichia coli بیان ژن آنزیم بنزیل فورمات دکربوکسیلاز در باکتری E.coli 113 131 729008 10.61882/JCT.16.2.113 FA مولود خیری دانش آموخته کارشناسی ارشد، مجتمع پدافند غیرعامل، دانشگاه صنعتی مالک اشتر، ایران علی اصغر دلدار استادیار، مجتمع پدافند غیرعامل، دانشگاه صنعتی مالک اشتر، ایران (aad.phd.gene@gmail.com) محمد‌جواد دهقان‌عصمت آبادی استادیار، مجتمع پدافند غیرعامل، دانشگاه صنعتی مالک اشتر، ایران (mohammad_dehghan@mut.ac.ir) نفیسه پورمهدی دانشجوی دکتری، مجتمع پدافند غیرعامل، دانشگاه صنعتی مالک اشتر، ایران Journal Article 2025 09 08 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). هدف: 1،2،4-بوتان تریول (BT)، یک ماده شیمیایی غیرطبیعی با کاربردهای دارویی و نظامی، از ویژگی‫های این ماده است. تولید زیستی BT هدف این تحقیق است، زیرا روش شیمیایی به‫دلیل محدودیت‌های کاتالیزوری و بازده پایین، اقتصادی نیست. تولید آنزیم بنزیل فورمات دکربوکسیلازبا هدف تکمیل مسیر آنزیمی سنتز زیستی BT می‌باشد که از زایلوز طی چهار واکنش آنزیمی انجام می‌شود و E. coli با دارا بودن دو آنزیم از این مسیر، با افزودن دو ژن دیگر، قادر به تکمیل آن خواهد بود. مواد و روش‫ها: به‫منظور ساخت سویه E. coli بیان‌کننده آنزیم بنزوئیل فرمات دکربوکسیلاز، ژن mdlC از P. putida تکثیر و در وکتورهای pBAD و pET28 کلون شد. پس از تایید کلونینگ با آزمایش‌های تاییدی، بیان پروتئین با ژل SDS-PAGE و عملکرد آنزیم مورد بررسی قرار گرفت. نتایج: پس از انتقال سازواره بیانی به سویه مناسب، حضور باند پروتئینی 56 کیلودالتونی در ژل SDS-PAGE، بیان ژن mdlC را تایید کرد. برای ارزیابی عملکرد آنزیم، وکتور نوترکیب pBAD.mdlC به سویه TOP10 ترنسفرم و تولید BT در محیط کشت با روش HPLC آنالیز شد. نتیجه‌گیری: بنزوئیل فرمات دکربوکسیلاز (BFD)، آنزیمی کلیدی در مسیر تولید بوتان تریول (BT) درE. coli است. در این مطالعه، ژن mdlC کد کننده BFD از P. putida تکثیر و در وکتورهای pBAD و pET28 کلون شد. سیستم بیانی pET28 به‫دلیل سهولت استفاده انتخاب شد و وکتور pBAD نیز به‫دلیل قابلیت القا مورد استفاده قرار گرفت. بیان پروتئین 56 کیلودالتونی با SDS-PAGE تایید و عملکرد آنزیم در تولید BT با HPLC بررسی شد.

https://jct.araku.ac.ir/article_729008_cac502fe07bd407069fcbc7868bca847.pdf

دانشگاه اراک سلول و بافت 2228-7035 16 2 2025 06 22 Assessing the Impact of Calcium Channel Blockers on Doxorubicin-Induced Cytotoxicity in MDA-MB-231 and MCF-7 Breast Cancer Cell Lines بررسی تاثیر مسدودکننده‌های کانال‌ کلسیم بر سمیت سلولی دوکسوروبیسین در رده‌ها‌ی سلولی سرطان پستان MDA-MB-231 و MCF-7 132 155 728842 10.61882/JCT.16.2.132 FA الهام قادری کارشناس‌ارشد، گروه علوم زیستی، دانشکده علوم پایه، دانشگاه کردستان، سنندج، ایران، elhamghaderi8@gmail.com راحله شاکری استادیار، گروه علوم زیستی، دانشکده علوم پایه، دانشگاه کردستان، سنندج، ایران، r.shakeri@uok.ac.ir 0000-0002-1048-1472 Journal Article 2025 09 02 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. هدف: هدف این مطالعه ارزیابی تاثیر ترکیبی دو داروی مسدودکنندهی کانال کلسیمی شامل آملودیپین و دیلتیازم بر سمیت دوکسوروبیسین در ردههای سلولی سرطان پستان است. مواد و روش‫ها: سلولهای MDA-MB-231 و MCF-7 با غلظتهای مختلف آملودیپین، دیلتیازم به‫صورت تکی و در ترکیب با دوکسوروبیسین به‫مدت 48 و 72 ساعت تیمار شدند. بقای سلولی با تست MTT بررسی شد. مقادیر IC50 از منحنی دوز-پاسخ، محاسبه شد. اثر ترکیبی داروها با محاسبه شاخص ترکیبی (CI) مورد بررسی قرار گرفت. فعالیت کاسپاز-3/7 به‫عنوان نشانگر القای آپوپتوزیس با روش سنجش آنزیمی مبتنی بر سوبسترای رنگزا (Ac-DEVE-pNA) در لیز سلولهای تیمارشده اندازهگیری شد. نتایج: نتایج نشان داد که آملودیپین و دیلتیازم با برابر با اثر مهاری بر تکثیر سلولهای سرطانی MDA-MB-231 و MCF-7 به‫صورت وابسته به غلظت و زمان دارند. ارزیابی فعالیت کاسپاز-3/7 در لیز سلول‌های تیمارشده با داروها نشان داد که آملودیپین باعث افزایش فعالیت کاسپاز-3/7 در هر ردهی سلولی می‌شود که نشان‌دهنده القای آپوپتوزیس است. در سلول‌های تیمارشده با دیلتیازم، فعالیت کاسپاز-3/7 تنها در سلول‌های MCF-7 مشاهده شد، در حالی‫که فعالیت کاسپاز-3/7 در سلول‌های MDA-MB-231 کم‫تر از کنترل بود که نتیجهگیری در این خصوص نیازمند مطالعات بیشتر است. مقدار CI ، در اکثر غلظتها بزرگ‌تر از یک بود که نشان‌دهنده تداخل آملودیپین و دیلتیازم با اثر سیتوتوکسیک دوکسوروبیسین در طیف وسیعی از غلظت‌ها است. نتیجهگیری: آملودیپین و دیلتیازم نه تنها قادر به القای مرگ سلولی در سلولهای سرطانی هستند، بلکه در غلظتهای پائین در اثر سیتوتوکسیک دوکسوروبیسین تداخل ایجاد میکنند. این نتایج اهمیت بررسی تعاملات دارویی مسدودکننده‌های کانال کلسیمی با شیمی‌درمانی را برجسته می‌سازد.

https://jct.araku.ac.ir/article_728842_a0fe627fdd196df164f06d2a5aae935e.pdf

دانشگاه اراک سلول و بافت 2228-7035 16 2 2025 06 22 Investigating the synergistic effect of chromosomal damage caused by the co-treatment with Vinblastine and Arsenic on HDF cell line using Micronucleus assay on Binucleated cells بررسی تاثیر هم افزایی صدمات کروموزومی ناشی از تیمار هم‫زمان وین بلاستین و آرسنیک بر رده سلولی HDF با استفاده از آزمون میکرونوکلئوس در سلول‫های دو هسته‫ای‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬ 156 169 728847 10.61882/JCT.16.2.156 FA خدیجه عبدی ولمی کارشناسی ارشد، گروه زیست شناسی، دانشکده علوم، دانشگاه فردوسی مشهد، مشهد، ایران، کدپستی 09177948974 azadeh.abdi1991@gmail.com 0009-0000-7318-5948 فرهنگ حداد دانشیار، گروه زیست شناسی، دانشکده علوم، دانشگاه فردوسی مشهد، مشهد، ایران، کدپستی 09177948974، haddad@um.ac.ir 0000-0002-5998-7183 مریم مقدم متین استاد، گروه زیست شناسی، دانشکده علوم، دانشگاه فردوسی مشهد، مشهد، ایران، کدپستی 09177948974، matin@um.ac.ir Journal Article 2025 09 02 Introduction: Heavy metal refers to any metal with relatively high density, with toxic effects even at ppb levels. Heavy metals are one of the main threats to human health. Arsenic (As) is a heavy metal found in our living environment due to its widespread use in industrial activities. Exposure to As and its absorption by different tissues may lead to damage to cardiovascular function, skin lesions, high blood pressure, and diabetes. As harmful effects on chromosome integrity have been proven a long time ago. It has also been classified as a potent carcinogen by the Environmental Protection Agency (EPA) and the International Agency for Research on Cancer (IARC). One of the damages caused by As is chromosome breakages. These damages are the result of this heavy metal's ability in producing free radicals and their interaction with the DNA molecule. The role of aneuploidy in cancer induction has been suggested in several investigations. Aneuploidy can lead to drastic changes in the genetic balance of normal cells, potentially forcing them towards cancer formation. Aims: Due to the close relation between chromosome instability and cancer induction and the ability of As to induce cancer, the question arises whether As can also play a role in chromosome instability? The design of this study is based on answering this question. Materials and methods: In this study, to analyze potential cell toxicity and chromosome abnormalities induced in different treatments, MTT and Micronucleus assay on Binucleated cells were used, respectively. Cell toxicity of different doses of As was investigated on HDF cells. According to the results of MTT, three non-toxic doses of 100, 200, and 300 ng/ml were selected. To be able to understand the effect of As treatment on chromosome instability induced by aneugenic agent, vinblastine was used. Vinblastine is classified as an aneugen in several studies. Co-treatment of three doses of As and three doses of 0.75, 1.00, and 2.00 ng/ml of vinblastine (Vin) on the induction of chromosome abnormalities was investigated on the HDF cell line using the Micronucleus assay on Binucleated cells. Results: According to the results of the MTT assay, cell toxicity induced by As at 24, 48, and 72 hours was at doses of 604.3, 397.3, and 228.6, respectively. As also led to chromosomal abnormalities. Mean frequency of micronucleated binucleates (BiMn) was 0.296, 0497, and 7.270 for 100, 200, and 300 ng/ml, respectively, compared to the frequency of BiMn in the control (0.078). Vinblastine treatment also significantly increased the frequency of BiMn in all doses used compared to the control. Finally, Co-treatment with three doses of vinblastine and three doses of As significantly increased chromosomal abnormalities to almost three to seven times compared to solo vinblastine treatment. Discussion: Results indicate that As can induce chromosomal abnormalities, which is represented by an increase in BiMn frequency. In addition, co-treatment with vinblastine and As leads to higher chromosomal abnormalities compared to solo treatment with vinblastine. Significant increase in frequency of BiMn in the later experiment suggests that As provides a suitable ground for chromosomal abnormalities induced by other aneugenic agents, here, vinblastine. This effect might be the result of direct interference of As with mechanisms of cell division control or inducing mutation in genes involved in such mechanisms through oxidative species, which leads to an increase in their errors. In both cases, the results of this study reveal an alternative mechanism for As-induced tumor formation. Conclusion: As not only involves cancer formation by direct damage to chromosomes but also provides a suitable ground for chromosomal mis-segregation in cell divisions, which is believed to be a main step towards cancer formation. هدف: آرسنیک یکی از فلزات سنگین موجود در محیط زندگی انسان است که تاثیرات خطرناک آن بر سلامت کروموزوم‫ها و همچنین القای سرطان مدت‫هاست که اثبات شده است. عمده صدمات آرسنیک به‫شکل شکستگی‫های کروموزومی می‫باشد. این صدمات ناشی از توان این فلز سنگین در ایجاد رادیکال‫های آزاد و به‫دنیال آن واکنش آن‫ها با مولکول DNA، است. به‫دلیل ارتباط نزدیک بین ناپایداری‫های تعدادی کروموزومی و سرطان، این سوال مطرح است که آیا القای سرطان توسط آرسنیک می‫تواند از طریق القای این نوع ناهنجاری‫ها نیز باشد؟ مواد و روش‏ها: در این مطالعه تاثیر کروموزومی هم‫زمان سه غلظت 100، 200 و 300 ng/ml از آرسنیک به‫همراه سه غلظت 75/0، 1 و 2 ng/ml از وین بلاستین، یک آنیوژن شناخته شده، بر القای صدمات کروموزومی در رده سلولی HDF با استفاده از آزمون میکرونوکلئوس در سلول‫های دو هسته‫ای مورد بررسی قرار گرفت. نتایج: تیمار با آرسنیک ضمن کاهش قدرت زیستایی سلول، باعث ایجاد صدمات کروموزومی شد. همچنین تیمار هم‫زمان با وین بلاستین میزان صدمات کروموزومی را به شکل معنی‫داری بین حدود سه تا هفت برابر در مقایسه با تیمار با وین بلاستین به‫تنهایی، افزایش داد. نتیجه‏گیری: نتایج بیانگر این است که تیمار با آرسنیک زمینه مناسب ایجاد اختلالات کروموزومی القایی با وین بلاستین را فراهم می‫نماید. این تاثیر ممکن است از طریق تداخل مستقیم آرسنیک با مکانیسم‫های کنترل صحت تقسیم سلولی بوده یا با ایجاد جهش در ژن‫های دخیل در این فرآیندها باشد.

https://jct.araku.ac.ir/article_728847_d21276265564a53c9c947cd53302182b.pdf

دانشگاه اراک سلول و بافت 2228-7035 16 2 2025 06 22 Investigation of the effect of hydroalcoholic extract of Terminalia chebula on survival and induction of apoptosis in cultured colorectal cancer cells on PCL/Gelatin nanofiber scaffold بررسی اثر عصاره هیدروالکلی هلیله سیاه روی بقا و القای آپوپتوزیس در سلول‌های سرطانی کولورکتال کشت شده بر داربست نانوفیبر PCL/Gelatin 170 184 728951 10.61882/JCT.16.2.170 FA علی بهنود دکترای عمومی دامپزشکی، گروه علوم پایه و پاتوبیولوژی، دانشکده دامپزشکی، دانشگاه رازی طیبه محمدی استادیار، گروه علوم پایه و پاتوبیولوژی، دانشکده دامپزشکی، دانشگاه رازی، کرمانشاه tymhd@razi.ac.ir مهرداد پویانمهر استادیار، گروه علوم پایه و پاتوبیولوژی، دانشکده دامپزشکی، دانشگاه رازی، کرمانشاه Journal Article 2025 09 06 Intruduction: Colorectal adenocarcinoma, a frequent malignancy of the large intestine, is often treated with chemotherapy. However, the significant toxicity of these drugs to healthy tissues poses a major challenge in achieving successful cancer treatment. Cell culture has enabled the development of in vitro models for drug and anti-cancer compound testing. However, traditional 2D monolayer cultures have limitations in studying cancer biology. To more accurately recapitulate in vivo cellular behavior, cells are now routinely cultured in three-dimensional environments, which closely resemble the native tissue in terms of morphology and gene expression profiles. Electrospun nanofibers, with their high surface area and porosity, closely mimic the extracellular matrix and promote cell-cell interactions. These scaffolds have revolutionized 3D cell culture, providing a powerful tool for evaluating anticancer drugs in a more physiologically relevant environment. In recent years, much research has focused on herbal medicines as potent drug candidates for inducing apoptosis. Terminalia chebula is a medicinal plant with a wide array of therapeutic applications, including anticancer effects. Although numerous studies have reported the growth of healthy tissues on nanofiber scaffolds, there is limited information regarding the growth of tumor cells on these scaffolds. Aim: This study investigated the anti-proliferative and apoptotic effects of Terminalia chebula extract on HT-29 colorectal cancer cells cultured in a PCL/Gelatin nanofiber scaffold model. Materials and Methods: Colorectal cancer cells (HT-29 cell line) were cultured in both conventional (2D) and nanofiber scaffold (3D) culture conditions. After 24 hours, the cells were treated with 10, 100, 200, and 500 μg/mL concentrations of the extract for 24 hours. Subsequently, the MTT assay was performed to determine cell viability and IC50. In addition, cells treated with the IC50 concentration were stained with acridine orange/ethidium bromide. The morphology of the scaffold and cell adhesion on it were also examined using scanning electron microscopy. The obtained data were statistically analyzed using SPSS software, one-way ANOVA, and Tukey's post-hoc test. Results: Our findings indicated that Scanning electron microscopy images revealed that the 3D nanofibrous scaffolds, both cell-free and seeded with HT-29 cells, exhibited desirable mechanical properties and adequate porosity. The cells cultured on these scaffolds demonstrated robust growth, proliferation, and a 3D morphology closely resembling in vivo conditions, indicating successful cell-scaffold interactions. The MTT assay revealed that cell viability decreased in a concentration-dependent manner following 24 hours of treatment with the extract. The IC50 of the extract was 500 μg/mL in 2D culture and 200 μg/mL in 3D culture after 24 hours of treatment. Based on the morphological assessment using acridine orange/ethidium bromide staining, it was determined that treatment with the extract induced apoptosis in HT-29 cancer cell line. Conclusion: Collectively, the findings of this study indicated that the hydroalcoholic extract of Terminalia chebula exhibits concentration-dependent cytotoxicity against cancer cells, with enhanced efficacy in a 3D culture model. This highlights the advantages of using nanofiber scaffolds to mimic the in vivo tumor microenvironment and to better understand the mechanisms of cancer progression. Future studies should further explore the potential of this model for drug discovery and mechanistic investigations. هدف: هلیله سیاه گیاهی با خاصیت ضد سرطانی است. هدف از این مطالعه، برررسی اثر عصاره آن روی سلولهای سرطانی در محیط کشت دو بعدی و سهبعدی است. مواد و روشها: عصاره هیدروالکلی هلیله سیاه با غلظتهای 0، 10، 100، 200 و 500 میکروگرم در میلیلیتر تهیه و اثر آن بر سلول‌های سرطانی روده بزرگ با روش کشت مرسوم (محیط کشت دوبعدی) و کشت بر داربست نانوفیبر PCL/Gelatin (محیط کشت سه بعدی) با کمک آزمون سنجش بقای سلولی (MTT) بعد از 24 ساعت تیمار بررسی شد. همچنین برای تعیین نوع مرگ سلولی تحت اثر عصاره، رنگ آمیزی آکریدین اورنج/اتیدیوم بروماید انجام شد. نتایج: عصاره در هر دو محیط کشت باعث مهار تکثیر سلولها شد. غلظت IC50 عصاره در محیط کشت دو بعدی 500 میکروگرم در میلیلیتر و در محیط کشت سه بعدی، 200 میکروگرم در میلیلیتر تعیین شد. عصاره باعث مرگ سلولی شد که بر اساس تغییرات مورفولوژیک سلول، آپوپتوزیس تشخیص داده شد. نتیجهگیری: عصاره هیدروالکلی هلیله سیاه توانست روی سلولهای سرطانی به‫صورت وابسته به غلظت اثر کشندگی داشته باشد و این اثر در شرایط کشت سه بعدی با استفاده از داربست نانوفیبر بیشتر از شرایط کشت دو بعدی مرسوم بود. کشت سلولهای سرطانی روی داربست نانوفیبری میتواند مدلی مناسب برای بررسی تاثیر عوامل ضد سرطان روی سلولها در شرایطی شبیه درون بدن فراهم کند.

https://jct.araku.ac.ir/article_728951_b800e4fa994d1ec3edc221cc02331036.pdf

دانشگاه اراک سلول و بافت 2228-7035 16 2 2025 06 22 Apoptosis-promoting effect of Augerin B in HT29 colorectal cancer cell line اثر پیش‌برنده آپوپتوزیس آگرین‫بی (Augerin B) در رده سلولی سرطان کولورکتال HT29‬‬‬‬‬‬‬ 185 199 728958 10.61882/JCT.16.2.185 FA الهه امینی استادیار، گروه علوم جانوری، دانشکده علوم زیستی، دانشگاه خوارزمی، تهران، ایران ابوالفضل شاکری دانشیار، گروه فارماکوگنوزی، دانشکده داروسازی، دانشگاه علوم پزشکی مشهد، ایران آذر شیخ الاسلامی استادیار، گروه پژوهشی بیولوژی سلولی و پزشکی بازساختی، جهاد دانشگاهی واحد استان قم، قم، ایران Journal Article 2025 09 07 Introduction: Colorectal cancer (CRC) is the second most commonly diagnosed malignant tumor in the world, with about 1,926,425 new cases estimated for 2022. The most therapeutic approaches for CRC include surgery, chemotherapy, targeted medicine, and radiation therapy. Each of these treatments exerts side effects such as fatigue, constipation, loss of appetite, and low blood cell counts. Natural products have provided valuable anticancer effects for years. An increasing number of studies have elucidated that plant-based natural compounds can act as an alternative chemotherapy option against CRC. It has been demonstrated that herbal bioactive compounds such as flavonoids, alkaloids, peptides, terpenoids, and steroids have the potential to effectively treat CRC. Sesquiterpenoid lactones, a subclass of terpenoid compounds, have been shown to induce anti-tumor effects. Aguerin B, a sesquiterpene lactone, exhibits cytotoxic effects against some types of tumors. Aims: The current study aims to investigate the effect of Aguerin B on HT29 colorectal cancer, and assess the molecular mechanism of Aguerin B in colorectal cancer by evaluating the type of cell death it induces and its effect on the expression of the tumor suppressor gene p53 in HT29 colorectal cancer cells. Materia and Methods: Colorectal cancer cells were purchased from the Iranian Biological Resource Center. HT29 cells were cultured in DMEM medium enriched with 10% fetal bovine serum and 1% antibiotics in 96-well plates. After 24 hours, the cells were treated with Aguerin B (variable concentrations from 1 to 8 μg/ml) for 24 and 48 hours. Cell viability was assessed using the MTT assay. Acridine orange-propidium iodide staining, caspase-3 and caspase-9 activity assays, using DCF-DA kits, were used to determine the type of cell death induced by Aguerin B. Additionally, qRT-PCR was used to evaluate p53 expression at the transcriptional level. Results: The findings demonstrated that Aguerin B exerts cytotoxic effects on colorectal cancer cells in a dose- and time-dependent manner, with an IC50 (Inhibitory Concentration) value of 4.6 μg/ml. Acridine orange-propidium iodide staining confirmed apoptosis induction at the IC50 concentration of Aguerin B. The cultivated activity of caspase-3 and -9, along with elevated ROS levels, indicated apoptosis induction via the mitochondrial pathway. Furthermore, the upregulation of p53 suggests the tumor-suppressive effect of Aguerin B in colorectal cancer cells. Discussion: Among the cell death mechanisms, the induction of apoptosis is a more profound mechanism of cell death mediated by anticancer bioactive compounds extracted from natural sources. Recent investigations have been focused on cancer therapeutic techniques that increase the apoptosis rate in tumor cells by disrupting mitochondrial biogenesis, leading to mitochondrial dysfunction. Caspases, as proteolytic enzymes, play a significant role as effector molecules in the apoptosis-induced cell death pathway. HT29 cells are sensitive to the chemotherapeutic drugs 5-fluorouracil and oxaliplatin, which are standard treatment options for colorectal cancer, and therefore were selected as chemotherapy-responsive colorectal cancer cells in this study. The results of this study exhibited that Aguerin B can induce cytotoxicity and anticancer effects in HT29 colorectal cancer cells in a dose- and time-dependent manner. This compound is also able to induce its properties through the recruitment of the intrinsic pathway, the caspase-dependent pathway in HT29 cells. Nevertheless, the role of p53 mutations in the pathogenesis of CRC has been recognized. The obtained data showed that Aguerin B can exert its anti-cancer potential. Previously, it was found that some natural products, including sesquiterpenoids, can also exert their suppressive effects against cancers by targeting the p53-MDM2 pathway. Conclusion: Given its potent cytotoxicity and apoptosis-inducing effects, Aguerin B, which can suppress HT29 cells, appears to be an effective compound for obstructing human colorectal cancer cells, warranting further investigation in the pre-clinical phase and clinical studies. هدف: در سالهای اخیر فراوردههای طبیعی اثرات ضد سرطان ارزشمندی ارائه کرده‫اند. جهت درک مکانیسم مولکولی اثر آگرین‫بی (Aguerin B) در سرطان کولورکتال، هدف این پژوهش بررسی نوع مرگ سلولی القا شده توسط آگرین‫بی و بیان فاکتور سرکوب‫گر تومور p53 در سلول‫های سرطان کلورکتال HT29 است. مواد و روش‏ها: سلول‫های سرطان کلورکتال در محیط کشت DMEM غنی شده با 10 درصد سرم جنین گاوی و 1 درصد آنتی بیوتیک پنی سیلین-استرپتومایسین درون پلیت 96 خانه کشت شد. پس از گذشت 24 ساعت، سلول‫ها توسط آگرین‫بی (غلظت‫های 1 تا 8 میکروگرم بر میلی‫لیتر) به‫مدت 24 و 48 ساعت تیمار شدند. آزمون MTT جهت ارزیابی بقای سلول‫ها، رنگ‫آمیزی اکریدین-اورنج-پروپودیوم-یداید، کیت کاسپاز 3، 9 و کیت DCF-DA جهت تعیین نوع مرگ سلولی القا شده توسط آگرین‫بی و از qRT-PCR جهت ارزیابی بیان p53 در سطح رونویسی استفاده شد. نتایج: یافتهها نشان داد آگرین‫بی به‫صورت وابسته به دوز و زمان موجب القای اثرات سیتوتوکسیک در سلول‫های سرطان کولورکتال می‫شود. غلظت 6/4 میکروگرم بر میلیلیتر به‫عنوان غلظت میانه مهاری (IC50) مشخص شد. رنگ آمیزی اکریدین-اورنج-پروپودیوم-یداید نشاندهنده القای آپوپتوزیس تحت تیمار با غلظت میانه مهاری آگرین‫بی بود. افزایش فعالیت کاسپاز 3 و 9 و افزایش میزان ROS، نشان‫دهنده القای آپوپتوزیس از طریق مسیر میتوکندری تحت تیمار با آگرین‫بی بود. به‫علاوه، افزایش بیان p53 نشان‫دهنده اثر سرکوبگر تومور تحت تیمار با آگرین‫بی بود. نتیجه‏گیری: با توجه به اعمال سمیت موثر و اثر القاکننده آپوپتوزیس، آگرین‫بی به‫عنوان یک ترکیب موثر در مهار سلول‫های سرطان کولورکتال انسانی HT29 در مطالعات پیش بالینی و بالینی پیشنهاد میشود.

https://jct.araku.ac.ir/article_728958_71440dc736ecfc60de15ee9e89d5d701.pdf

دانشگاه اراک سلول و بافت 2228-7035 16 2 2025 06 22 Investigating the genetic stability of clones obtained from the micropropagation of lateral buds of watermelon under in vitro culture conditions بررسی ثبات ژنتیکی کلون‌های حاصل از ریزازدیادی جوانه‌های جانبی هندوانه در شرایط کشت درون‌شیشه‌‌ 200 227 729009 10.61882/JCT.16.2.200 FA شبنم مازندرانی کارشناسی ارشد، گروه بیوتکنولوژی، دانشکده کشاورزی و منابع طبیعی، دانشگاه بین‌المللی امام خمینی (ره)، قزوین، ایران، shabnam.mazandarani@yahoo.com قاسمعلی گروسی استاد، گروه بیوتکنولوژی، دانشکده کشاورزی و منابع طبیعی، دانشگاه بین‌المللی امام خمینی (ره)، قزوین، ایران، garoosi@eng.ikiu.ac.ir رحیم حداد دانشیار، گروه بیوتکنولوژی، دانشکده کشاورزی و منابع طبیعی، دانشگاه بین‌المللی امام خمینی (ره)، قزوین، ایران، r.haddad@eng.ikiu.ac.ir Journal Article 2025 09 08 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). هدف: هدف این پژوهش ریزازدیادی رقم جابانی هندوانه از طریق کشت جوانه جانبی و بررسی تغییرات ژنتیکی احتمالی گیاه‫چه‌ها پس از چهار نسل باززایی است. مواد و روش‌ها: در این تحقیق، جوانه‌های جانبی پس از رشد گیاه مادری، در محیط کشت MS با غلظت‌های مختلف IAA، BAP و TDZ منتقل و ریزازدیادی به‫مدت چهار نسل ادامه یافت. در ادامه تاثیر غلظت‌های منتخب هورمونی و نسل‌های مختلف واکشت روی شباهت/تفاوت ژنتیکی گیاه‫چه‌های انتخابی با گیاه شاهد (مادری) توسط نشانگر ISSR موردبررسی قرار گرفت. نتایج: نتایج حاصل از بررسی تاثیر هورمونها روی افزایش تعداد جوانه جانبی در نسل‌های مختلف نشان داد که به‌طورکلی سه غلظت 0 mg/L IAA و 1 mg/L BAP، 1 mg/L IAA و 2 mg/L BAP و 2 mg/L IAA و 2 mg/L BAP به‫ترتیب با 33/2، 83/3 و 17/2 نوساقه در هر ریزنمونه بیشترین تاثیر را روی ازدیاد تعداد جوانه‌های جانبی بعد از چهار نسل داشتند. در بررسی تغییرات ژنتیکی با استفاده از 5 نشانگر ISSR، 60 باند تولید شد که 3/43 درصد آن‌ها چندشکلی بودند. بیشترین تعداد باند (17 باند) مربوط به آغازگر UBC-864 و کمترین (9 باند) مربوط به P2 بود. بیشترین جایگاه‌های چندشکلی در آغازگر UBC-808 مشاهده شد. در میان تیمارها مورد بررسی، تیمار 1 mg/L IAA و 2 mg/L BAP در نسل 3 ریزازدیادی با 9 جایگاه چندشکلی بیشترین و تیمار 0 mg/L IAA با 1 mg/L BAP در نسل 1 ریزازدیادی با 2 جایگاه چندشکلی کمترین تعداد چندشکلی را نشان دادند. نتیجهگیری: یافته‌های مطالعه کنونی نشان داد که نشانگرهای ISSR به‌طور موثری می‌توانند برای بررسی ثبات یا تنوع ژنتیکی ایجادشده در هندوانه پس از چند نسل واکشت در شرایط درون شیشه استفاده شوند.

https://jct.araku.ac.ir/article_729009_57c5989be756bc19bb6e6dc67d5c71db.pdf