CRISPR/Cas9-mediated knockout of HO-1 decreased the proliferation and migration of T47D cells and increased cisplatin-induced apoptosis: an in vitro study.

Breast cancer is the most common type of neoplasm and the second cause of cancer-related death in women. Despite the development of novel therapeutic strategies and improved the clinical outcomes, the mortality rate for breast cancer is still high. Therefore, development of a new modality, particularly based on knocking out key genes, is under focus of investigation. Heme oxygenase-1 (HO-1) deregulation has been associated with various neoplasms-related behaviors of many types of tumor cells including breast cancer. In the current study, in order to evaluate the role of the HO-1 gene in breast cancer, we utilized the CRISPR/Cas9 technology to knock out HO-1 gene in T47D breast cancer cell line and studied its potential therapeutic effects in vitro. The cell proliferation and their sensitivity to Cisplatin were determined by CCK-8 kit. In addition, the apoptosis and the migratory potential of the cells were evaluated using Hoechst staining, and Transwell/Scratch methods, respectively. Our findings revealed that HO-1 suppression significantly reduced the proliferation ability of T47D cells (P < 0.001). Moreover, sensitivity to Cisplatin-induced toxicity increased significantly in KO-T47D cells compared to the control T47D cells. Furthermore, our findings indicated that Cisplatin-induced apoptosis increased in the KO-T47D cells. Moreover, the migratory capability of KO-T47D cells was abolished significantly (P < 0.001) as determined by Transwell migration assay. In a nutshell, our findings strongly suggest that HO-1 involved in breast cancer progression and metastasis and chemotherapy resistance. However, further comprehensive studies are required to clarify the precise role of the HO-1 gene on breast cancer cells.

An In vitro Study on the Protective Effect of Melatonin on Human Sperm Parameters Treated by Cadmium.

Background: Male infertility account for nearly 50% of infertility cases. Cadmium is regarded as a well-known toxic metal for industrial applications; high amounts of cadmium in the human body can result in chronic toxicity. Melatonin as a free radical scavenger has anti-inflammatory, and even anti-cancer and antiapoptotic functions. Aim: In this work, we evaluated the protective effect of melatonin on human sperm parameters treated by cadmium. Study Setting and Design: This was an experimental study carried out from May to December 2019. Materials and Methods: A total of 41 fresh semen samples were collected from fertile men and were divided into 4 groups: (1) control, (2) sperm +25 Nm cd, (3) sperm +25 nM cd +0.1 mM melatonin,(4) sperm +0.1 mM melatonin treated for 60 min. In all groups, semen analysis was performed for motility, viability and DNA fragmentation index (DFI). Statistical Analysis: The groups were compared using the ANOVA test. Results: The group treated with cadmium showed a significant decrease in rapid and slow motility, and survival rate compared with the control group (P < 0.05). However, the degree of DFI and sperm with non-progressive motility in the group treated with cadmium had a significant increase compared to the control (P < 0.05). The use of melatonin significantly improved sperm parameters such as motility, survival rate and decreased sperm DFI with non-progressive motility. Conclusions: The use of melatonin reduces the amount of cadmium damage in human sperm in vitro.

Effect of astaxanthin and melatonin on cell viability and DNA damage in human breast cancer cell lines.

BACKGROUND: Astaxanthin is a xanthophyll pigment found in algae and marine animals, having strong anti-oxidative, anti-tumoral, and anti-inflammatory effects. Additionally, melatonin has shown inhibitory effects on the growth of human breast cancer cells. The aim of the present study was to evaluate the effect of astaxanthin and the combined effects of astaxanthin and melatonin on breast cancer cells and the non-tumoral breast cell line. MATERIALS AND METHODS: The human breast cancer cell lines, T47D and MDA-MB-231, and non-tumorigenic cell line MCF 10A were treated and compared to astaxanthin, melatonin, and co-administration of these two compounds. Cell viability, apoptosis induction, Bcl-2 protein expression, and DNA damage were measured by MTT assay, acridine orange/ethidium bromide (AO/EB) staining, immunocytochemistry, and comet assay. RESULTS: Astaxanthin at lower doses than melatonin reduced cell viability and Bcl2 expression, induced apoptosis and DNA damage in MDA-MB-231 and T47D. Meanwhile, the effects of astaxanthin on cell cytotoxicity, apoptosis, and DNA damage in MCF10A cells are insignificant compared to MDA-MB-231 and T47D. Moreover, the results indicated that astaxanthin in T47D cells caused more cell death compared to MDA-MB-231 cells. Astaxanthin induced cell death on breast cancer cells and without cell cytotoxicity for non-cancerous cells. CONCLUSION: Furthermore, the presence of astaxanthin increased the function of melatonin-induced cell death in breast cancer cells.

Dimethyl fumarate prevents cytotoxicity and apoptosis mediated by oxidative stress in human adipose-derived mesenchymal stem cells.

BACKGROUND: The poor survival rate and undesirable homing of transplanted stem cells are the major challenges in stem cell therapy. Addressing the challenge would improve the therapeutic efficacy of these cells. Dimethyl fumarate (DMF) is an anti-inflammatory drug that exerts its effects through the activation of the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway. Therefore, its cytoprotective effects on human adipose-derived MSCs (hASCs) against various oxidative stresses have been investigated in this study. METHODS AND RESULTS: hASCs were cultured with different concentrations of DMF to evaluate the cytotoxicity of DMF on hASCs using Cell Counting Kit-8 (CCK-8). Besides, the migration ability of the cells after DMF treatment was evaluated using the Transwell method. Furthermore, the expression of HO-1 and NQO-1 was determined using RT-PCR. The cytoprotective effects of DMF on hASCs against the oxidative stress caused by H2O2 and Ultra Violet (UV) were evaluated by assessing cell proliferation and apoptosis. Our results demonstrated that under oxidative stress conditions induced by H2O2 and UV, DMF increased the survival rate and proliferation of the cells and prevented apoptosis. Moreover, the expression of HO-1 and NQO-1 was upregulated in hASCs pretreated with DMF which confirms the activation of the Nrf2 pathway. However, DMF significantly decreased migration in hADSCs (P < 0.0001). CONCLUSION: Our findings indicate that DMF enhances the proliferation capability and viability of hASCs and prevents their apoptosis in harsh stressful microenvironments. However, the applicability of DMF as a cytoprotective factor for the augmentation of hASCs requires in-depth preclinical and clinical studies.

Effects of melatonin on the toxicity and proliferation of human anaplastic thyroid cancer cell line.

BACKGROUND: Thyroid carcinoma is the most common endocrine malignancy and anaplastic thyroid carcinoma (ATC) is a rare but most aggressive cancer. Melatonin has enhanced or induced apoptosis in many different cancer cells, however, there has not been any study on the effects of melatonin in the treatment of ATC. In this study, we examined the effect of melatonin on cytotoxicity in the human ATC cell line. MATERIALS AND METHODS: Cultured ATC cells were treated at melatonin concentrations 0.6, 1, 4, 16, 28 mM for 24 h. The MTT assay was performed to examine cell viability. Cytotoxicity was assayed with the determination of lactic dehydrogenase (LDH) activity. Apoptosis was detected by acridine orange/ethidium bromide and Hoechst 33342 staining. Giemsa staining is considered for evaluating the morphological changes of ATC cells. The reproductive ability of cells to form a colony was evaluated by the clonogenic assay. RESULTS: Results showed that melatonin could significantly decrease cell viability and the lowest cell viability was observed at 28 mM, 10.26 % ± 0.858 versus control. Similar results were obtained when analyzing LDH activity. The highest LDH levels were observed at 16 and 28 mM (546.08 ± 4.66, 577.82 ± 3.14 munit/mL versus control) that confirmed the occurrence of late apoptosis. The clonogenic assay showed that cells at the high concentration of melatonin (16 and 28 mM) don't enable to form the colony that approved the occurrence of reproductive death. CONCLUSION: Our results showed a dose-dependent cytotoxic effect of melatonin on ATC cells that significantly decreased cell viability and induced cell reproductive death at the concentration greater than 1 mM and findings suggested that MLT might be useful as an adjuvant in ATC therapy.

Anti-proliferative and apoptotic effects of hull-less pumpkin extract on human papillary thyroid carcinoma cell line.

Papillary thyroid carcinoma (PTC) is one of the most common cancers of the endocrine system. Previous studies have shown that the extract of hull-less pumpkin seed (HLPS) has a significant anti-cancer effect. The aim of this study was to evaluate the effect of this plant extract on the proliferation of PTC cells. In this study, an extract of this plant was prepared by soxhlet extraction method and analyzed by Gas Chromatography-Mass Spectrometry. The cytotoxicity of PTX and plant extract was investigated using the methylthiazol tetrazolium (MTT) method. For careful investigation of morphological alteration, we used hematoxylin and eosin and Giemsa stinging. Based on MTT assay test, the IC50 value of paclitaxel (PTX) was significantly less than the hydro-alcoholic extract of HLPS at all of the incubation time. Our results of histological staining showed that HLPS and PTX induced significant morphological alteration in the PTC cultured cell that consistent with cell death. Comparing the groups treated by PTX or HLPS with control group showed significant differences. It seems that HLPS extract has an apparent effect on treatment of PTC, at least in laboratory condition, albeit for realistic decision about the effect of HLPS on PTC, more molecular investigations are necessary.

Oxytocin can decrease germ cells apoptotic index in testis under acute ischemia reperfusion in a rat model.

UNLABELLED: Ischemia reperfusion (IR) is the main pathology of torsion of testis and it is a common urologic emergency. There is some evidence that shows oxytocin (OT) plays role in ischemia reperfusion. OBJECTIVE: To evaluate this hypothesis that OT can decrease germ cell apoptotic index in testis under acute ischemia reperfusion in a rat model. MATERIALS AND METHODS: 20 adult rats were randomly divided into four groups: Control, IR, OT and IR+ OT (OTA). Testicular ischemia was achieved by 720° torsion of the left testis for 2 hr. Then, torsion was removed and reperfusion was performed. Immediately after induction of reperfusion 0.03 µg/kg OT were administered intraperitoneally to the IR+ OT. Three hours after surgery left testis was removed and evaluations were made by Johnson's score, ELISA, immunohistochemistry and histomorphometry for study of maturity of spermatogenesis, endocrine profiles, apoptosis and quantitative studies, respectively. RESULTS: The results showed in addition tissue edema and congestion, a significant reduced in Johnson's score were detected in IR group in comparison with controls (p=0.01), and apoptotic index increased significantly (p=0.001). Administration of OT in OT+IR group, increased Johnson's score but it was not statistically significant. Germinal epithelium thickness was increased significantly (p=0.03), although apoptotic index decreased significantly in comparison with the IR group (p=0.04). However there was not significant difference in serum levels of testosterone, FSH and LH in none of groups (p=0.07). CONCLUSION: These results suggested that OT can decrease apoptotic index and improves complication of acute ischemic reperfusion in testis in a rat model.

Morphological identification of cell death in dorsal root ganglion neurons following peripheral nerve injury and repair in adult rat.

BACKGROUND: Axotomy causes sensory neuronal loss. Reconnection of proximal and distal nerve ends by surgical repair improves neuronal survival. It is important to know the morphology of primary sensory neurons after the surgical repair of their peripheral processes. METHODS: Animals (male Wistar rats) were exposed to models of sciatic nerve transection, direct epineurial suture repair of sciatic nerve, autograft repair of sciatic nerve, and sham operated. After 1 and 12 weeks of the surgery, the number of L5 dorsal root ganglion (DRG) and ultrastructure of L4-L5 DRG neurons was evaluated by fluorescence and electron microscopy, respectively. RESULTS: Nerve transection caused sensory neuronal loss and direct epineurial suture but no autograft repair method decreased it. Evaluation of morphology of the neurons showed classic features of apoptosis as well as destructive changes of cytoplasmic organelles such as mitochondria, rough endoplasmic reticulum and Golgi apparatus in primary sensory neurons. These nuclear and cytoplasmic changes in primary sensory neurons were observed after the surgical nerve repair too. CONCLUSION: The present study implies that the following peripheral nerve transection apoptosis as well as cytoplasmic cell death contributes to neuronal cell death and reconnection of proximal and distal nerve ends does not prevent these processes.