THE EFFECT OF N- ACETYLCYSTEINE ON THE NEUROTOXICITY OF SEVOFLURANE IN DEVELOPING HIPPOCAMPUS CELLS.

Sevoflurane, a common pediatric anesthetic, has been linked to neurodegeneration, raising safety concerns. This study explored N-acetylcysteine's protective potential against sevoflurane-induced neurotoxicity in rat hippocampi. Four groups were examined: Control: Received 6hours of 3l/min gas (air and 30% O2) and intraperitoneal saline. NAC: Received 6hours of 3l/min gas and 150mg/kg NAC intraperitoneally. Sev: Exposed to 6hours of 3l/min gas and 3% sevoflurane. Sev+NAC: Received 6hours of 3l/min gas, 3% sevoflurane, and 150mg/kg NAC. Protein levels of NRF-2, NLRP3, IL-1ß, caspase-1, Beclin 1, p62, LC3A, and apoptosis markers were assessed. Sevoflurane and NAC alone reduced autophagy, while Sev+NAC group maintained autophagy levels. Sev group had elevated NRF-2, NLRP3, pNRF2, Caspase-1, and IL-1ß, which were reduced in Sev+NAC. Apoptosis was higher in Sev, but Sev+NAC showed reduced apoptosis compared to the control. In summary, sevoflurane induced neurotoxicity in developing hippocampus, which was mitigated by N-acetylcysteine administration.

Apoptosis-inducing, anti-angiogenic and anti-migratory effects of a dinuclear Pd(II) complex on breast cancer: A promising novel compound.

Because of the high mortality and morbidity rate of breast cancer, successful management of the disease requires synthesis of novel compounds. To this end, ongoing attempts to create new candidates include synthesis of multinuclear metal complexes. The high DNA binding affinity and cytotoxic activity of these complexes makes them promising as breast cancer treatments. This study investigated anti-growth/cytotoxic effect of the dinuclear Pd(II) complex on breast cancer cell lines (MCF-7, MDA-MB-231) using various methods of staining, flow cytometry, and immunoblotting. The study conducted colony formation, invasion, and migration assays were to assess the effect of the complex on metastasis. Increased caspase-3/7 levels and positive annexin V staining were observed in both cell lines, proving apoptosis. Altered TNFR1 and TRADD expression with caspase-8 cleavage followed by BCL-2 inactivation with loss of mitochondrial membrane potential confirmed the presence of apoptosis in MCF-7 and MDA-MB-231, regardless of p53 expression status. The results implied anti-migration properties. Finally, the study used the CAM assay to assess antiangiogenic properties and showed that the complex inhibited angiogenesis. The study concluded the dinuclear Pd(II) complex warrants further in vivo experiments to show its potential in the treatment of breast cancer.

Comparison of the anti-cancer activity of 5-aminolevulinic acid-mediated photodynamic therapy after continuous wave and pulse irradiation in different histological types of canine mammary sarcoma tumors.

Canine mammary sarcoma tumors (CMST) are the most aggressive tumors with poor prognosis in dogs. Due to inadequate treatment options for CMST, recent studies have focused on alternative treatment strategies. We previously determined the optimized protocol of 5-ALA-based photodynamic therapy (PDT) in canine liposarcoma. However, its molecular mechanisms in the treatment of different histological types of CMST remain unclear.In this context, we, for the first time, assessed 5-aminolevulinic acid (5-ALA)-PDT-mediated anti-cancer activity and its molecular mechanism after continuous wave (CW) and pulse radiation (PR) on three different histological types (liposarcoma, chondrosarcoma, and osteosarcoma) of CMST cells by WST-1, Annexin V, ROS, acridine orange/propidium iodide staining, RT-PCR, and western blot analysis.Our findings showed that 5-ALA/PDT significantly suppressed the proliferation of CMST cells (p < 0.01) and induced apoptosis via increased ROS level and overexpression of Caspase-9 and Caspase-3 mRNA and cleaved protein levels in especially liposarcoma and chondrosarcoma cells following CW and PR irradiation at 9 J/cm2. However, the response of CMST cells to 5-ALA was different upon CW and PR irradiation due to differences in their origin.Collectively, our findings provided the first evidence that 5-ALA-based PDT could be used as an alternative treatment strategy, especially liposarcoma and chondrosarcoma. However, further in vitro and in vivo studies are required to elucidate the underlying molecular mechanism of the efficacy of 5-ALA in CMST cells at the molecular level.

Evidence for heterogeneity in response to treatment in mammary tumors of dogs as happens in humans.

Tumors are formed by various clones developed over a long time. This gives rise to a heterogeneous nature. This heterogeneity is the hardest challenge in the treatment of cancers because it is the main reason for drug resistance. This is a well-known fact in human cancer. Therefore, we have reasoned that if the tumor heterogeneity in canine mammary gland tumors (CMGTs) could be shown by an ex vivo assay, which will be used first time in veterinary oncology practice, this could be used further in clinics. To achieve this, twenty-six patients were included in the study. Tumor tissues were obtained from animals during routine surgery. Tumor cells were isolated and seeded ex vivo. The cells were exposed to anticancer drugs that are clinically used. Seven days after the treatment, chemosensitivity has luminometrically been assayed by ATP-tumor chemosensitivity assay (ATP-TCA). It has clearly been shown that all the tumor tissues have responded to treatment differently, implying that heterogeneity exists in mammary tumors. There has also been found that there was a weak to moderate statistically significant correlation between tumor size and drug index. However, there has been no correlation between drug index and metastasis to lymph nodes. Hyperplasic areas had relatively higher PCNA values. The results of our study demonstrate the heterogeneity in responses to in vitro drugs. Clinical trials based on test results and follow-up studies with large numbers of animals are needed to prove that such chemotherapeutic activity assessment tests can be clinically useful in predicting drug responses in CMGTs.

The response of the canine mammary simple carcinoma and carcinosarcoma cells to 5-aminolaevulinic acid-based photodynamic therapy: An in vitro study.

BACKGROUNDS: Canine mammary gland tumors (CMGTs) are heterogeneous tumors and share many similar features with human breast cancer. Despite the improvement of current treatment options, new treatment modalities are required to effectively kill tumor cells without general toxicity in the treatment of CMGTs. Photodynamic therapy (PDT) is a promising method for cancer treatment. However, there is a limited study evaluating the therapeutic efficacy of PDT in the treatment of CMGTs. METHODS: In this context, we, for the first time, investigated the therapeutic potential of 5-aminolaevulinic acid (5-ALA) mediated PDT at 6 and 12 J/cm2 in two different subtypes [Tubulopapillary carcinoma (TPC) and carcinosarcoma (CS)] cells via different molecular analysis. The cytotoxic effects of 5-ALA/PDT on these cells were analyzed by intracellular PpIX level, WST-1 and ROS analysis. Furthermore, the underlying moleculer mechanism of 5-ALA/PDT mediated apoptotic effects on TPC and CS cells were evaluated Annexin V, AO/PI, RT-PCR and western blot analysis. RESULTS: The 5-ALA/PDT treatment upon irradiation considerably inhibited the viability of both TPC and CS cells (p<0.01) and caused apoptotic death through elevated ROS levels, the activation of Caspase-9, and Caspase-3, and the overexpression of Bax. However, the response of TPC and CS cells to 5-ALA/PDT was different. CONCLUSIONS: Our preliminary in vitro findings provide novel insights into the molecular mechanisms underlying 5-ALA/PDT mediated apoptosis in both TPC and CS cells. However, the therapeutic response of CMGT cells to 5-ALA/PDT is limited.

Effects of combined administration of doxorubicin and chloroquine on lung pathology in mice with solid Ehrlich ascites carcinoma.

Combined use of a chemotherapeutic agent and an autophagy inhibitor is a novel cancer treatment strategy. We investigated the effects of chloroquine (CQ) on lung pathology caused by both solid Ehrlich ascites carcinoma (EAC) and doxorubicin (DXR). A control group and eight experimental groups of adult female mice were inoculated subcutaneously with 2.5 × 106 EAC cells. DXR (1.5 mg/kg and 3 mg/kg) and CQ (25 mg/kg and 50 mg/kg) alone or in combination were injected intraperitoneally on days 2, 7 and 12 following inoculation with EAC cells. Lung tissue samples were examined using immunohistochemistry (IHC) for endothelial (eNOS), inducible nitric oxide synthase (iNOS) and neutrophil gelatinase-associated lipocalin (NGAL). Serum catalase (CAT), glutathione peroxidase (GPx), superoxide dismutase (SOD) and malondialdehyde (MDA) levels were measured using ELISA. We found decreased levels of iNOS and eNOS in the groups that received 1.5 mg/kg DXR alone and in combination with 25 mg/kg and 50 mg/kg CQ. Combined administration of DXR and CQ partially prevented disruption of alveolar structure. Levels of antioxidant enzymes and MDA were lower in all treated groups; the greatest reduction was observed in mice that received the combination of 25 mg/kg CQ + 1.5 mg/kg DXR. Levels of NGAL were elevated in all treated groups. We found that CQ ameliorated both EAC and DOX induced lung pathology in female mice with solid EAC by reducing oxidative stress.

Palladium (II) complex and thalidomide intercept angiogenic signaling via targeting FAK/Src and Erk/Akt/PLCγ dependent autophagy pathways in human umbilical vein endothelial cells.

The current study assessed the effects of the thalidomide and palladium (II) saccharinate complex of terpyridine on the suppression of angiogenesis-mediated cell proliferation. The viability was assessed after treatment with palladium (II) complex (1.56-100 µM) and thalidomide (0.1-400 µM) alone by using ATP assay for 48 h. Palladium (II) complex was found to inhibit growth statistically significant in a dose-dependent manner in HUVECs and promoted PARP-1 cleavage through the production of ROS. On the other hand, thalidomide did not cause any significant change in cell viability. Moreover, cell death was observed to be manifested as late apoptosis due to Annexin V/SYTOX staining after palladium (II) complex treatment however, thalidomide did not demonstrate similar results. Thalidomide and palladium (II) complex also suppressed HUVEC migration and capillary-like structure tube formation in vitro in a time-dependent manner. Palladium (II) complex (5 mg/ml) treatment showed a strong antiangiogenic effect similar to positive control thalidomide (5 mg/ml) and successfully disrupted the vasculature and reduced the thickness of the vessels compared to control (agar). Furthermore, suppression of autophagy enhanced the cell death and anti-angiogenic effect of thalidomide and palladium (II) complex. We also showed that being treated with thalidomide and palladium (II) complex inhibited phosphorylation of the signaling regulators downstream of the VEGFR2. These results provide evidence for the regulation of endothelial cell functions that are relevant to angiogenesis through the suppression of the FAK/Src/Akt/ERK1/2 signaling pathway. Our results also indicate that PLC-γ1 phosphorylation leads to activation of p-Akt and p-Erk1/2 which cause stimulation on cell proliferation at lower doses. Hence, we demonstrated that palladium (II) and thalidomide can induce cell death via the Erk/Akt/PLCγ signaling pathway and that this pathway might be a novel mechanism.

Hydrogen Sulphide and Nitric Oxide Cooperate in Cardioprotection Against Ischemia/Reperfusion Injury in Isolated Rat Heart.

BACKGROUND/AIM: This study was designed to provide further evidence for the interactions between hydrogen sulfide (H2S) and nitric oxide (NO) in ischemia/reperfusion (I/R) injury. MATERIALS AND METHODS: Rat hearts were studied with the Langendorff technique using the H2S donor sodium hydrosulfide (NaHS, 40 µM) and the cystathionine gamma-lyase (CTH or CSE) inhibitor DL-propargylglycine (PAG, 1 mM). NO synthase inhibitor L-NG-nitroarginine methyl ester (L-NAME, 30 mg/kg, 7 days) was administered before the isolation. The hearts were homogenized for biochemical and molecular analysis. RESULTS: NaHS reversed I/R-induced cardiac performance impairment, increased tissue nitric oxide production and decreased tissue markers for cardiac injury, while L-NAME inhibited these effects. The expression of CTH was increased with PAG, which was suppressed by L-NAME. CONCLUSION: H2S and NO increase each other's production suggesting their interaction and cooperation in cardioprotection against I/R injury.

The effect of high mobility group box-1 protein on cerebral edema, blood-brain barrier, oxidative stress and apoptosis in an experimental traumatic brain injury model.

Traumatic brain injury (TBI) is one of the important reason of morbidity and mortality. While the primary injury due to mechanical impact is unavoidable, the secondary injury which is formed as a result of primary injury and thought to occur due to neuroinflammation in the forefront can be prevented and by this way mortality and morbidity can be reduced. High mobility group box-1 (HMGB1) is a protein that triggers the neuroinflammatory process by being released from the nucleus of necrotic tissues after primary injury. The aim of this study is to investigate the effects of HMGB1 on its receptors TLR4 and RAGE, cerebral edema, blood-brain barrier, oxidative stress and apoptosis causing secondary damage in an experimental traumatic brain injury model. Weighing between 280-320 g, 10 to 12 weeks-old, a total of 30 adult male Sprague-Dawley rats were used for the experiments. The rats were randomly assigned to 3 groups: 1) Control, 2) TBI and 3) TBI + ethyl pyruvate group (n = 10 per group). Right parietal cortical contusion was made by using a weight-dropping TBI method. Brain samples were harvested from pericontusional area at 24 h after TBI. HMGB1, TLR4, RAGE, occludin, claudin-5, ZO-1 levels are investigated by western blot analyses and immunohistochemistry examinations. HMGB-1, TLR4 and RAGE expressions increased after TBI. Major tight junction proteins in the blood-brain barrier: occludin, claudin-5 and ZO-1 expressions decreased after TBI. Brain edema increased after TBI. Also, proapoptotic bax and active caspase 3 expressions increased, antiapoptotic bcl-2 levels decreased after TBI. Total oxidant status and oxidative stress increased, total antioxidant status decreased after TBI. HMGB-1 protein plays a key role in the pathophysiology of traumatic brain injury.

Chloroquine Used in Combination with Chemotherapy Synergistically Suppresses Growth and Angiogenesis In Vitro and In Vivo.

BACKGROUND: The inhibition of autophagy using pharmacological inhibitors such as chloroquine may be an effective strategy to overcome chemotherapy or resistance to anti-angiogenic therapy. MATERIALS AND METHODS: The cytotoxic effect of doxorubicin (0.1-1 µM), chloroquine (0.25-32 µM) and their combination were investigated by employing ATP assay in human umbilical vein endothelial cells (HUVECs). The effect of doxorubicin and chloroquine combination was also measured using tube formation assay on Matrigel. The anti-angiogenic activities of doxorubicin (2.5 µg/pellet) and chloroquine (15 µg/pellet), their combination, and standards (50 µg/pellet) were tested in vivo using the chick embryo chorioallantoic membrane (CAM) assay. RESULTS: The combination of doxorubicin and chloroquine significantly had a stronger anti-angiogenic effect than the positive control (±)-thalidomide and doxorubicin alone in the CAM assay and in vitro tube-formation assay. CONCLUSION: Chloroquine enhanced the anti-angiogenic effect of doxorubicin on CAM at the tested concentrations.

Anticancer effect of a novel palladium-saccharinate complex of terpyridine by inducing apoptosis on Ehrlich ascites carcinoma (EAC) in Balb-C mice.

BACKGROUND/AIM: [Pd(sac)(terpy)](sac)•4H2O (sac=saccharinate and terpy=2,2':6',2"-terpyridine) is newly-synthesized palladium(II) (Pd) complex. We investigated the antiproliferative and apoptotic effects of this complex on Ehrlich ascites carcinoma (EAC). MATERIALS AND METHODS: EAC cells were administered to 33 Balb/c mice. Mice were divided randomly into four groups: control, cisplatin, Pd(II) complex and paclitaxel. Control group animals received 0.9% NaCl; other groups received treatments cisplatin, Pd(II) complex and paclitaxel on days 7 and 12. At day 14, animals were sacrificed. Expression of active caspase-3, p53 and proliferating cell nuclear antigen (PCNA) was investigated and apoptosis was evaluated by terminal deoxynucleotidyltransferase (TdT)-mediated nick-end labelling (TUNEL) technique. RESULTS: Expression of p53 and PCNA were found to be decreased (p<0.0001), cells with active caspase-3 and TUNEL-positive cells were found to be increased (p<0.0001) in all treatment groups. CONCLUSION: Like cisplatin and paclitaxel, this Pd(II) complex has a strong anticancer activity against EAC by inducing apoptosis and suppressing proliferation in vivo.

Apoptosis-inducing effect of a palladium(II) saccharinate complex of terpyridine on human breast cancer cells in vitro and in vivo.

The anti-growth effect of a palladium(II) complex-[PdCl(terpy)](sac)·2H2O] (sac=saccharinate, and terpy=2,2':6',2″-terpyridine)-was tested against human breast cancer cell lines, MCF-7 and MDA-MB-231. Anti-growth effect was assayed by the MTT and ATP viability assays in vitro and then confirmed on Balb/c mice in vivo. The mode of cell death was determined by both histological and biochemical methods. The Pd(II) complex had anti-growth effect on a dose dependent manner in vitro and in vivo. The cells died by apoptosis as evidenced by the pyknotic nucleus, cleavage of poly-(ADP-ribose) polymerase (PARP) and induction of active caspase-3. These results suggest that the palladium(II) saccharinate complex of terpyridine represents a potentially active novel complex for the breast cancer treatment, thus warrants further studies.