Activation of epidermal growth factor receptors in triple-negative breast cancer cells by morphine; analysis through Raman spectroscopy and machine learning.

Triple negative breast cancer (TNBC) is a very aggressive form of breast cancer, and the analgesic drug morphine has been shown to promote the proliferation of TNBC cells. This article investigates whether morphine causes activation of epidermal growth factor receptors (EGFR), the roles of µ-opioid and EGFR receptors on TNBC cell proliferation and migration. While examining the changes with molecular techniques, we also aimed to investigate the analysis ability of Raman spectroscopy and machine learning-based approach. Effects of morphine on the proliferation and migration of MDA.MB.231 cells were evaluated by MTT and scratch wound-healing tests, respectively. Morphine-induced phosphorylation of the EGFR was analyzed by western blotting in the presence and absence of µ-receptor antagonist naltrexone and the EGFR-tyrosine kinase inhibitor gefitinib. Morphine-induced EGFR phosphorylation and cell migration were significantly inhibited by pretreatments with both naltrexone and gefitinib; however, morphine-increased cell proliferation was inhibited only by naltrexone. While morphine-induced changes were observed in the Raman scatterings of the cells, the inhibitory effect of naltrexone was analyzed with similarity to the control group. Principal component analysis (PCA) of the Raman confirmed the epidermal growth factor (EGF)-like effect of morphine and was inhibited by naltrexone and partly by gefitinib pretreatments. Our in vitro results suggest that combining morphine with an EGFR inhibitor or a peripherally acting opioidergic receptor antagonist may be a good strategy for pain relief without triggering cancer proliferation and migration in TNBC patients. In addition, our results demonstrated the feasibility of the Raman spectroscopy and machine learning-based approach as an effective method to investigate the effects of agents in cancer cells without the need for complex and time-consuming sample preparation. The support vector machine (SVM) with linear kernel automatically classified the effects of drugs on cancer cells with ∼95% accuracy.

Investigation of the effects of umbilical cord-derived mesenchymal stem cells and curcumin on Achilles tendon healing - can they act synergistically?

BACKGROUND: It is known that curcumin and umbilical cord-derived mesenchymal stem cells (UC-MSCs) positively affect experi-mental tendon injury healing. This study investigated individual effects and potential synergistic effects of using curcumin and UC-MSCs alone and together. METHODS: Eighty female Wistar albino rats were randomly divided into five groups: Control, curcumin, sesame oil, MSCs, and Curcumin+MSCs groups. In all rats, punch tendon defect was created in both right and left Achilles tendons. While no additional treatment was applied to the control group, curcumin, sesame oil used as a solvent for curcumin, MSCs, and MSCs and curcumin com-bination were applied locally to the injury site, respectively, in the other groups. Curcumin was solved in sesame oil before application. In each group, half of the animals were euthanized in the post-operative 2nd week while the other half were euthanized in the post-operative 4th week. The right Achilles was used for biomechanical testing, while the left Achilles was used for histological evaluation and immunohistochemical analysis of type I, Type III collagen, and tenomodulin. RESULTS: Histologically, significant improvement was observed in the curcumin, MSCs, and Curcumin+ MSCs groups compared to the control Group in the 2nd week. In the 2nd and 4th weeks, Type III collagen was significantly increased in the curcumin group com-pared to the control group. In week 4, tenomodulin increased significantly in the curcumin and MSCs groups compared to the control group. Tendon tensile strength increased significantly in MSCs and Curcumin+MSCs groups compared to the control group in the 4th week. No superiority was observed between the treatment groups regarding their positive effects on recovery. CONCLUSION: Locally used curcumin and UC-MSCs showed positive effects that were not superior to each other in the healing of injury caused by a punch in the Achilles tendons of rats. However, synergistic effects on healing were not observed when they were applied together.

Food-Grade Physically Unclonable Functions.

Counterfeit products in the pharmaceutical and food industries have posed an overwhelmingly increasing threat to the health of individuals and societies. An effective approach to prevent counterfeiting is the attachment of security labels directly on drugs and food products. This approach requires the development of security labels composed of safely digestible materials. In this study, we present the fabrication of security labels entirely based on the use of food-grade materials. The key idea proposed in this study is the exploitation of food-grade corn starch (CS) as an encoding material based on the microscopic dimensions, particulate structure, and adsorbent characteristics. The strong adsorption of a food colorant, erythrosine B (ErB), onto CS results in fluorescent CS@ErB microparticles. Randomly positioned CS@ErB particles can be obtained simply by spin-coating from aqueous solutions of tuned concentrations followed by transfer to an edible gelatin film. The optical and fluorescence microscopy images of randomly positioned particles are then used to construct keys for a physically unclonable function (PUF)-based security label. The performance of PUFs evaluated by uniformity, uniqueness, and randomness analysis demonstrates the strong promise of this platform. The biocompatibility of the fabricated PUFs is confirmed with assays using murine fibroblast cells. The extremely low-cost and sustainable security primitives fabricated from off-the-shelf food materials offer new routes in the fight against counterfeiting.

Self-Healing of Biocompatible Superhydrophobic Coatings: The Interplay of the Size and Loading of Particles.

The broad application potential of superhydrophobic coatings is limited by the usage of environment-threatening materials and poor durability. The nature-inspired design and fabrication of self-healing coatings is a promising approach for addressing these issues. In this study, we report a fluorine-free and biocompatible superhydrophobic coating that can be thermally healed after abrasion. The coating is composed of silica nanoparticles and carnauba wax, and the self-healing is based on surface enrichment of wax in analogy to the wax secretion in plant leaves. The coating not only exhibits fast self-healing, just in 1 min under moderate heating, but also displays increased water repellency and thermal stability after healing. The rapid self-healing ability of the coating is attributed to the relatively low melting point of carnauba wax and its migration to the surface of the hydrophilic silica nanoparticles. The dependence of self-healing on the size and loading of particles provides insights into the process. Furthermore, the coating exhibits high levels of biocompatibility where the viability of fibroblast L929 cells was ∼90%. The presented approach and insights provide valuable guidelines in the design and fabrication of self-healing superhydrophobic coatings.

Morphine counteracts the effects of paclitaxel in triple-negative breast cancer cells.

Background & objectives: Several studies have provided evidence that opioids may play a role in cancer recurrence and metastasis. Multiple research data indicate that morphine can act as a proliferative or suppressive agent on tumour cells depending on the applied concentration. Therefore, this study was aimed to investigate whether the presence of clinically relevant concentrations of morphine has any effect on the efficacy of paclitaxel, a widely used chemotherapeutic drug, on the viability and apoptosis of human triple-negative breast cancer cell line. Methods: MDA.MB.231 cells were treated with paclitaxel in the presence or absence of morphine and examined for cell proliferation by the MTT assay. In addition, the effect of morphine on paclitaxel-induced apoptosis was investigated by flow cytometric assay and by the ratio of Bax/Bcl-2 mRNA expression levels with quantitative real-time (qRT)-PCR. Results: Morphine significantly increased the proliferation of breast cancer cells at low concentrations (0.1-2.5 µM) but higher concentrations showed cytotoxic effect. Pre-treatment with 0.1 or 1 µM of morphine decreased the paclitaxel-induced cytotoxicity, the proportion of apoptotic cell, and the ratio of Bax/Bcl-2 mRNA expressions. Interpretation & conclusions: Our data suggest that morphine promotes breast cancer cell viability at clinically relevant plasma concentrations and reduces the apoptotic effect of paclitaxel. This interaction may be very important in clinical settings; however, more studies are needed to explore the plausible mechanisms of interaction and to correlate such findings through in vivo animal studies as well as clinically.

Bone marrow-derived mesenchymal stem cells alleviate paclitaxel-induced mechanical allodynia in rats.

Anticancer drug paclitaxel (PTX) frequently causes painful peripheral neuropathy; however, no medication has been shown to be effective in the treatment of this debilitating side effect. We aimed to investigate the efficacy of two different doses of allogeneic bone marrow-derived mesenchymal stem cells (BM-MSCs) on PTX-induced mechanical allodynia and spinal cytokine levels and their localization to target tissues such as the spinal cord and sciatic nerve. After the development of mechanical allodynia with repeated PTX administration, two different doses of rat BM-MSCs, low or high (1 × 106 -5 × 106 ), were transplanted into rats and the evaluation continued for 30 days. Interleukin (IL)-1ß, tumor necrosis factor (TNF)-α, and IL-10 levels in spinal cord samples of animals were analyzed by enzyme-linked immunosorbent assay. PTX-induced mechanical allodynia was relieved significantly 15 days after the transplantation of high-dose of BM-MSCs. Both MSCs doses were effective in alleviating allodynia, but the onset of effect was earlier with the high dose. High-dose of BM-MSCs significantly decreased spinal IL-1ß and TNF-α levels compared to the PTX group. Fluorescent dye-labeled BM-MSCs were observed much more frequently in the sciatic nerve and spinal cord samples of the high-dose BM-MSCs transplanted group than in the low-dose group animals. In conclusion, we found that the antiallodynic effects of BM-MSCs appeared earlier when high-dose of cells were administered. We think that other mechanisms may play a role in the effects of MSCs, besides localization to damaged tissues and reducing spinal inflammatory cytokine levels. We show that BM-MSCs can be a novel approach in PTX-induced mechanical allodynia.

Melatonin has an inhibitory effect on MCF-7 and MDA-MB-231 human breast cancer cell lines by inducing autophagy and apoptosis.

The goal of this work was to see how melatonin affected Bax and Bcl-2 expression, as well as apoptosis and autophagy, in MCF-7 and MDA-MB-231 breast cancer cell lines, which have distinct hormonal sensitivities. In this study, to investigate the IC50 value of melatonin, varied melatonin concentrations were administered to MCF-7 and MDA-MB-231 breast cancer cell lines. Moreover, cytotoxic activities were analyzed through MTT analysis. Five subgroups were created for both cell lines: control, IC50-MeL, hIC50-MeL, DMSO1, and DMSO2. To evaluate the apoptotic effect of melatonin, immunofluorescence staining methods of TUNEL, Bax, and Bcl-2 were used, and to examine the effects of autophagy, immunofluorescence staining methods of Beclin-1, LC3, and p62 were used. In vitro results revealed upregulation of the expression of TUNEL and Bax in both MCF-7 and MDA-MB-231 cell lines regarding dose and time, but downregulation of Bcl-2 expression. Moreover, autophagy results were consistent with in vitro apoptosis results in both MCF-7 and MDA-MB-231 cell lines. We determined that the expressions of the autophagy markers Beclin-1, LC3, and p62 were increased. Our findings indicate that treatment of breast cancer cells with melatonin increased the inhibitory effect of melatonin on cell growth through both apoptosis and autophagy in vitro. Consequently, it was concluded that melatonin might adjust the expression balance of markers that have a role in cell death mechanisms and significantly promote these mechanisms. Therefore, melatonin can inhibit the growth of breast cancer cells by inducing cell death.

Indomethacin prevents TGF-ß-induced epithelial-to-mesenchymal transition in pancreatic cancer cells; evidence by Raman spectroscopy.

Pancreatic ductal adenocarcinoma (PDAC) has a very low survival rate due to the late detection and poor response to chemotherapy. Epithelial-to-mesenchymal transition (EMT) is considered an important step in tumor progression with regard to invasion and metastasis, and Transforming Growth Factor-beta (TGF-ß) signaling has been shown to play an important role in EMT. Therefore, we aimed to investigate whether indomethacin, an anti-inflammatory and analgesic drug, has any effect on TGF-ß-induced EMT in pancreatic cancer cell line and analyze the changes in their molecular structures by Raman spectroscopy and other molecular techniques. Indomethacin treated Panc-1 cells were analyzed with Raman spectroscopy, quantitative polymerase chain reaction and immunofluorescence techniques after the induction of EMT with TGF-ß. The exposure of Panc-1 cells to TGF-ß resulted in characteristic morphological alterations of EMT, and indomethacin inhibits TGF-ß-induced EMT through up-regulation of E-cadherin and down-regulation of N-cadherin and Snail expressions. Raman spectroscopy supported by principal component analysis (PCA) confirmed the effects of both TGF-ß and indomethacin. Raman spectra were further analyzed using the PCA-assisted vector machine algorithm and it was seen that the data could be classified with 97.6% accuracy. Our results suggest that indomethacin may have a significant effect on PDAC metastasis, and Raman spectroscopy was able to probe EMT-related changes and the efficacy of indomethacin in a short time and without the need for specific reagents compared to other molecular techniques.

The role of autophagy in paclitaxel and cremophor induced damage to rat testis.

The anticancer drug, paclitaxel (PTX), is used to treat a variety of solid tumors, but its effects on normal tissues remain unclear. We investigated the effects of different doses of PTX and its vehicle, cremophor EL, on testis using histochemical, immunohistochemical and biochemical methods. We used 30 adult Wistar albino rats divided randomly into five groups: physiological saline was administered to the control group; the sham 8 group received 8 mg/kg cremophor EL; the sham 16 group received 16 mg/kg cremophor EL; 8 mg/kg PTX was administered to the PTX 8 group; and the PTX 16 group received 16 mg/kg PTX. The cremophore content in PTX groups was the same as in the sham group. All treatments were injected intraperitoneally (i.p.) once/week for 4 weeks. Tissue samples were excised 24 h after the last injection. Tissue sections were prepared and hematoxylin and eosin staining was performed to assess testicular morphology. Expression of Beclin-1, LC3A/B and P62 were assessed using immunohistochemistry. Serum and tissue testosterone levels were determined using ELISA. Light microscopy revealed seminiferous tubule damage, irregularities in germinal epithelium and decreased seminiferous tubule diameter in the PTX treated groups. The immunoreactivity of Beclin-1, LC3A/B, and P62 was increased significantly in the PTX groups compared to the control group. Cremophor EL alone damaged the testis, although not as much as PTX. PTX caused significant damage to testicular tissue and increased autophagy of spermatogenic cells; cremophore EL also may play a role in this effect.

The Effect of Cyclosporine A on Proteins Controlling Intracellular Calcium Concentration in Breast Cancer Cells.

Cyclosporine A (CsA) is an immunosuppressive drug commonly used to prevent autoimmune diseases. At the same time, CsA is a calcineurin (CaN) inhibitor. It affects the intracellular calcium signaling pathway. The effect of CsA on breast cancer cells, MDA-MB-231, plasma membrane calcium pump 1 (PMCA1), calmodulin (CaM), calcineurin (CaN), and cMyc, which are proteins that affect calcium signaling, were investigated. CsA inhibited the proliferation of MDA-MB-231 cells but did not affect the migration of the cells. After 24 h of incubation, CsA suppressed the PMCA1 protein, which pumps intracellular calcium out of the cell. At the same time, calcium started to accumulate inside the cell and CaM protein was expressed, while PMCA1 was suppressed. The CaN protein was suppressed 72 h after the administration of CsA, but the cMyc protein was expressed. Interestingly, 24 h incubation when the PMCA1 protein is down-regulated after the duration of time, the cMyc protein is also down-regulated. Although the indirect effect of CaN and cMyc is known, this relationship between PMCA1 and cMyc was not known. As a result, it has been shown that CsA affects the PMCA pump by disrupting the intracellular calcium pathway in breast cancer cells.

The effect of erythropoietin and umbilical cord-derived mesenchymal stem cells on nerve regeneration in rats with sciatic nerve injury.

OBJECTIVE: We aimed to investigate the effects of umbilical cord-derived mesenchymal stem cells and erythropoietin on nerve regeneration in the sciatic nerve 'crush injury' in a rat model. METHODS: Experimental animals were randomly divided into 5 groups: Crush Injury, Sham, Crush Injury + Erythropoietin, Crush Injury + Mesenchymal Stem Cell, Crush Injury + Erythropoietin + Mesenchymal Stem Cell groups. Crush injury made with bulldog clamp. Mesencyhmal stem cells delivered by enjection locally. Erythropoietin administered by intraperitoneally. On the 0th, 14th and 28th days, all groups underwent a sciatic functional index test. On 28th day, sciatic nerves were harvested and histopathological appearance, axon number and axon diameter of the sciatic nerves were evaluated with Oil Red O staining. Immunoreactivity of nerve growth factor, neurofilament-H and caspase-3 were determined by immunofluorescence staining in nerve tissue. RESULTS: In histopathological examination, axons and nerve bundles exhibiting normal nerve architecture in the Sham group. Crush Injury + Mesenchymal Stem Cell group has similar histological appearance to the Sham group. The number of axons were higher in the Mesenchymal Stem Cell groups compared to the Crush Injury group. Nerve growth factor immunoreactivity intensity was significantly lower in Crush Injury + Mesenchymal Stem Cell group compared to Crush Injury group. Neurofilament-H density was higher in the treatment groups when compared to the Crush Injury group. CONCLUSIONS: In this study, it was found that umbilical cord-derived mesenchymal stem cells and erythropoietin treatments effects positively regeneration of crush injury caused by bulldog clamp in the sciatic nerve of rats.

Clinical plasma concentration of vinpocetine does not affect osteogenic differentiation of mesenchymal stem cells.

AIM: Vinpocetine (Vin) has long been used as a medicine to treat cerebrovascular disorders and as a dietary supplement to improve cognitive functions. Previous studies have revealed that the transcription factor nuclear factor kappa B (NF-κB) activity plays an important role in osteogenic differentiation of mesenchymal stem cells (MSC). Vin inhibits NF-κB-dependent inflammatory responses; however, the effect of Vin on the osteogenic differentiation of MSCs has not been reported. In this study, we aimed to the investigate effect of Vin on the osteogenic differentiation of rat bone marrow-derived MSCs (BMSCs). METHODS: We treated BMSCs with clinical plasma (0.17 µM) or higher concentrations (5 and 20 µM) of Vin with no significant effect on the cell viability. Alizarin Red S and alkaline phosphatase (ALP) stainings were used to evaluate mineralizations on days 14 and 21. Moreover, expressions of target genes were detected using qRT-PCR analysis. RESULTS: Osteogenic differentiation of BMSCs did not significantly change with Vin's clinical plasma concentration, but significantly decreased with higher concentrations. Calcium mineralization, ALP staining and mRNA gene expressions of Runx2 and ALP were decreased significantly with high concentrations of Vin, paticularly on day 21. CONCLUSION: Our in vitro findings suggest that clinically relevant concentration of Vin seems safe to use in elderly patients with respect to osteoporosis. On the other hand, Vin at high concentrations appears to be harmful to bone homeostasis.

Raman spectroscopy: A novel experimental approach to evaluating cisplatin induced tissue damage.

The aim of this work is to clarify the effect of curcumin and beta-carotene on cisplatin-induced tissue damage and to demonstrate the potential of Raman spectroscopy to detect tissue changes consistent with liver and kidney histopathology as a potential diagnostic adjunct. In the study, 56 Wistar albino female rats were used and randomly divided into 7 groups (n:8). Sham group received only sesame oil; Cisplatin group, received a single dose injection of cisplatin; Beta-carotene group, treated with beta-carotene orally; Cisplatin + Beta-carotene group, pretreated with beta-carotene 30 min prior to the cisplatin injection, then received cisplatin; Curcumin group, orally treated with curcumin; Cisplatin + Curcumin group, pretreated with curcumin 30min prior to the cisplatin injection, then received cisplatin. The second application was performed 1 week after the first application. One of the liver and kidney tissues was taken to 10% form for histopathological examinations and the others were taken to -80 °C for raman spectroscopy. Received sections were hematoxylin-eosin stained. The avidin-biotin peroxidase method was used for to investigate anti-TNF-α and IL1-ß activities. TUNEL method was applied to determine apoptotic cells. According to our histopathological findings, beta-carotene and especially curcumin have been found to possess hepatorenal protective activities. These datas were supported by the microscopic damage scores. Although some of these findings were observed in both the cisplatin + curcumin and cisplatin + beta-carotene groups, the incidence and severity of histopathological lesions were less than the cisplatin group. Both immunohistochemical studies and Raman spectroscopy results consistent with histopathological examination of hematoxylen-eosin stained sections. Raman spectroscopy represents a suitable tool to provide insights into structural factors involved in the mechanisms underlying antitumor effects of platinum drug.

Anti-microsporidial effect of thymoquinone on Encephalitozoon intestinalis infection in vitro

Objective: To evaluate the anti-microsporidial effects of the active component of Nigella sativa seeds, thymoquinone, against Encephalitozoon intestinalis using an in vitro model. Methods: Anti-microsporidial effect of thymoquinone against Encephalitozoon intestinalis was evaluated by using various concentrations of thymoquinone (0, 1, 5, 10, 15, 20, 30, 35, and 40 μM) and sterile dimethyl sulfoxide. Real time PCR was used to evaluate the inhibitory effects of thymoquinone on the life cycle of Encephalitozoon intestinalis. Results: The cytotoxic effect of thymoquinone on HEK293 cell line was observed with 30, 35, and 40 μM concentrations of thymoquinone after 24, 48, and 72 hours of incubation. It was observed that 10, 15, 20, and 30 μM concentrations of thymoquinone decreased the spore density compared with the control; however, it was significant only at 30 μM. Conclusions: Thymoquinone shows potent anti-microsporidial effects against Encephalitozoon intestinalis in the in vitro model;however, the toxic concentrations of thymoquinone are also toxic to the host cells.

Ephedrine enhances the antinociceptive effect of dexmedetomidine in mice.

OBJECTIVES: Dexmedetomidine, a highly selective alpha-2-adrenoceptor agonist, was recently introduced into clinical practice for its sedative and analgesic properties. The purpose of this study was to evaluate whether the psychostimulant drug ephedrine has any effect on dexmedetomidine-induced antinociception and locomotor inhibitor activity in mice in acute application. METHODS: In both sexes of swiss albino mice; antinociception was assessed with hot-plate test and the locomotor, exploratory activities were assessed with holed open field test. The animals were received; saline + saline, ephedrine (10 mg/kg) + saline, saline + dexmedetomidine (15 µg/kg) and ephedrine (10 mg/kg) + dexmedetomidine (15 µg/kg), intraperitoneally, 30 min before hot plate or holed open field tests. RESULTS: In the hot plate test in mice, co-administration of 15 µg/kg dexmedetomidine with 10 mg/kg ephedrine intraperitoneally not only enhanced, but also prolonged the duration of antinociception induced by dexmedetomidine. At the same time, the locomotor inhibitory effect of dexmedetomidine was counteracted by ephedrine. CONCLUSION: We concluded that the combined administration of dexmedetomidine with ephedrine may have beneficial effects in the treatment of pain without causing sedation, which limits the use of dexmedetomidine as an analgesic in humans.

Effects of agmatine on nicotine-evoked behavioral responses in rats.

Agmatine [2-(4-aminobutyl)guanidine], is a novel endogenous ligand at alpha2-adrenoceptors, imidazoline and N-methyl-D-asparate receptors, as well as a nitric oxide synthase inhibitor. The present study tested whether agmatine (5-40 mg/kg, sc) modulated the locomotor, sensitizing, and discriminative stimulus effects of nicotine in male Wistar rats. Agmatine (10-40 mg/kg) affected neither the basal locomotor activity, nor the nicotine (0.4 mg/kg, sc)-evoked hyperactivation. A challenge with saline or nicotine (0.4 mg/kg, sc) on day 10 to rats treated repeatedly (for 5 days) with nicotine (0.4 mg/kg, sc; and exposed to experimental chambers), resulted in the expression of nicotine-evoked conditioned hyperlocomotor response or behavioral sensitization. Given on day 10, agmatine at a dose of 40 mg/kg (but not 20 mg/kg) attenuated nicotine-induced conditioned hyperactivity. However, when this dose was administered to the nicotine-sensitized rats, agmatine failed to alter the effect of the challenge dose of nicotine. In rats trained to discriminate nicotine (0.4 mg/kg, sc) from saline in a two-lever water-reinforced fixed ratio 10 task, agmatine (20 or 40 mg/kg) did not substitute for the training dose of nicotine. In combination studies, pretreatment with agmatine (5-40 mg/kg) did not affect the nicotine (0.4 mg/kg) discrimination and the fixed dose of agmatine (20 mg/kg) did not change the effects of the lower doses of nicotine (0.05-0.2 mg/kg). Our pharmacological analyses indicate that agmatine does not affect the locomotor, sensitizing, or subjective effects of nicotine. However, these data do show an inhibitory effect of agmatine over the expression of nicotine-induced conditioned hyperlocomotion.