Inhibition of ALA dehydratase activity in heme biosynthesis reduces cytoglobin expression which is related to the proliferation and viability of keloid fibroblasts.

The aim of this study was to analyze the effect of heme synthesis inhibition on cytoglobin expression and its correlation with keloid fibroblast viability and proliferation. The study was conducted on primary culture of keloid fibroblasts. Heme synthesis in keloid fibroblasts was inhibited using succinyl acetone. We measured amino levulinic acid dehydratase (ALAD) enzyme activity using a colorimetric method; cytoglobin mRNA expression using qRT-PCR, cytoglobin protein expression using ELISA and immunocytochemistry, fibroblast viability using the MTT test; and fibroblast proliferation using BrdU test. The results showed that the ALAD enzyme activity level was lower in the keloid fibroblasts treated with succinyl-acetone (SA, 1, 2.5, and 5 mM) than in the control. The cytoglobin mRNA and protein expressions level were significantly lower in the keloid fibroblasts cultured with 2.5 mM and 5 mM SA than in the control and 1 mM SA. The viability and proliferation of the keloid fibroblasts decreased when the SA concentration was increased. In conclusion, the use of succinyl acetone at a concentration of 1; 2.5; and 5 mM caused decrease ALAD enzyme activity which indicated the inhibition of the heme synthesis. Inhibition of heme synthesis can affect cytoglobin expression, which correlates with the viability and proliferation of keloid fibroblasts.

Effect of allopurinol on oxidative stress and hypoxic adaptation response during surgical correction of tetralogy of fallot.

AIM: to analyze the role of allopurinol in reducing the ischemia-reperfusion injury, and to confirm the HIF-1 binding activity changes during the surgical correction of tetralogy of fallot (TF). METHODS: randomized, double-blind experimental study on patients undergoing surgical correction of TF in the Integrated Cardiovascular Services, Cipto Mangunkusumo Hospital from September 2009-May 2010. Patients were randomly divided into two groups. The first group was given 10 mg/kg body weight of allopurinol 3 times before undergoing operation (n=13) and the other group was given placebo (n=13). Tissue specimen from right ventricular muscle were taken for measurement of reactive oxygen species (ROS) expression and blood specimens from intra-coronary sinus for measurement of TNF-, superoxide dismutase (SOD), and malondialdehyde (MDA). RESULTS: cardiomyocytes expressing ROS in placebo group increased (41.37±29.29%; 42.61±22.82% and 53.81±25.76%), while in allopurinol group decreased (44.68±19.79%, 56.87±15.50%, and 47.98±22.52%). Concentration of TNF- tend to decrease in allopurinol group, while it tended to increase in the placebo goup. Concentration of SOD increased in the allopurinol group, while in the placebo group there were no significant changes. Concentration of MDA was highly increased in the placebo group (1.75 pmol/mg; 2.37 pmol/mg; 2.72 pmol/mg; 4.82 pmol/mg), but statistically it was not significant. On the other hand, there was no significant change of MDA concentration in the allopurinol group. Expression of HIF-1 in TF patients decreased significantly (p=0.026) from pre ischemic phase to ischemic phase, but it increased in the reperfusion phase (0.95 OD/mg protein, 0.52 OD/mg protein, 0.9 OD/mg protein). CONCLUSION: pre-surgical oral allopurinol treatment reduced ischemia-reperfusion injury during TF surgical correction, as indicated by the decrease in the number of cardiomyocytes expressing ROS, reducing TNF-, SOD, and MDA concentration in blood. There was a decrease in HIF-1 concentration in cardiomyocytes of the right ventricle during ischemic phase.

Conjugation of Cetuximab - Puromycin and Its Target-Specific Effect on Triple Negative Breast Cancer Cell Lines.

Cancer is life-threatening disease and being global health problems. Chemotherapy is one of the most used therapy for cancer since many years ago. Chemotherapy is also toxic for normal cell, not specific to the target cells. Consequently, chemotherapy has various side effects. Monoclonal antibody (MAb) has been developed for specific therapy which only has killing effect in cancer cells, but the survival rate of most MAbs around 20%. Therefore, in clinical practice, MAbs administration should combine with chemotherapeutic agents. For effectiveness of therapy and to minimalize adverse effects, anticancer agent with selective cytotoxic effect on target cells is needed, the immunotoxin. OBJECTIVE: This study introduces a novel approach to conjugate monoclonal antibody (Cetuximab) and toxin (Puromycin), in order to selectively inhibit proliferation of triple negative breast cancer (TNBC) and to enhance the efficacy of MAb in target cells killing. METHODS: Cetuximab was conjugated with Puromycin using a linker, i.e SATP (Succinimidyl-acetylthiopropionate) and tested on triple negative breast cancer cell lines (MDA-MB-231) which expressed EGFR (epidermal growth factor receptor). Cetuximab is MAb which targets EGFR. MCF-7 was used as control cells since it has low or no EGFR expression. Cell counting were conducted as viability assay at 24 hours, 48 hours, and 72 hours after treatment. RESULTS: The results showed significant reduction of live cells number in Conjugate 20 µg/mL cultured in MDA-MB-231 compared to MCF-7 after 24 hours, 48 hours, and 72 hours incubation. In all time period of incubation, significant reduction of MDA-MB-231 live cells number was also observed in Conjugate 20 µg/mL compared to Cetuximab 20 µg/mL. CONCLUSION: Synthesized conjugate showed its target-specific effect in TNBC and improved the efficacy of Cetuximab on TNBC. In the future, this conjugate can be a potential anticancer therapy in treating triple-negative breast cancer.

Resistance Mechanism of Acute Myeloid Leukemia Cells Against Daunorubicin and Cytarabine: A Literature Review.

Acute myeloid leukemia (AML) is a hematological malignancy commonly found in adult patients. Low overall survival and resistance to therapy are the main issues in AML. The first line of treatment for AML chemotherapy is the induction phase, namely, the phase to induce remission by administering a combination of daunorubicin (DNR) for three days followed by administration of cytarabine (Ara-C) with continuous infusion for seven days, which is referred to as "3 + 7." Such induction therapy has been the standard therapy for AML for the last four decades. This review article is made to discuss daunorubicin and cytarabine from their chemical structure, pharmacodynamics, pharmacokinetics, and mechanisms of resistance in AML.

Gastric ulcers induced by systemic hypoxia.

AIM: to assess the effect of systemic hypoxia on gastric mucosa and the activation of stress-responsive transcription factors induced by hypoxia. METHODS: in this experimental study, rats were allocated to control and experimental groups. The experimental group was divided into subgroups and subjected to hypoxia conditions for 1, 7, 14 or 21 days. Afterwards, histopathological evaluation and study of the protein expression of the gastric mucosa were performed. RESULTS: the results showed that longer exposure to hypoxic conditions leads to more severe gastric ulceration. Twenty-four hours after induction, 60% of rats had developed gastric ulcers. Seven days after induction, 80% of rats developed gastric ulcers. In the 14-day and 21-day hypoxia conditions, epithelialization (a sign of gastric ulcer healing) was observed. Evaluation of the average ulcer depth on the day of treatment showed that the greatest depth was on day 7, and the shallowest was on day 21 of treatment. Western blot analyses demonstrated that systemic hypoxia resulted in the expression of heat shock factor (HSF) and heat shock protein 70 (HSP-70), which were highest on day 7 and then regressed gradually. In control, HSF-1 and HSP-70 were not detected by Western blot analysis in the control group (normoxia). CONCLUSION: in this study, systemic hypoxia caused gastric ulcers, and during the time of exposure to hypoxia, an adaptation process in the form of gastric epithelialization occurred in the rats. This development of gastric lesions was in line with the expression pattern of HSF-1 HIF-1 and HSP-70.

Structural and morphological changes in rat ventricular myocardium induced by chronic systemic hypoxi.

AIM: to explore the effects of chronic systemic hypoxia on myocardial structure and morphology. In addition, the goal of present study is to develop a hypoxia-induced heart failure model in rats. METHODS: Sprague-Dawley male rats, weighing 220-250 g at the time of recruitment were randomly allocated into 7 groups (n = 4 per group), the control normoxia group was exposed to room air, while the hypoxia groups were caged in a plexiglas hypoxic chamber (8% O2 and 92% N2) for 28 days. Structural and morphological changes of ventricular myocardium were determined at day 28, while blood gas parameters were measured at day 1, 3, 7, 14, 21, and 28. RESULTS: histopathologic and morphologic evaluation showed massive hypertrophy accompanied by damage of the intercalated disk (ID) structure, angiogenesis, necrosis, fibrosis, and apoptosis as a hallmark of ventricular remodeling. At the end of treatment, there were increases of LV (2.79 vs 3.71) and RV (1.72 vs 2.54) wall thicknesses, and also in hypertrophy index (from 3.19 to 5.74). Blood gas analysis revealed metabolic acidosis compensated by respiratory alkalosis. There was an observed decrease of blood gas parameters in hypoxia group compared to control group: PO2 (24.7 vs 96.4 mm Hg), PCO2 (18.2 vs 40.4 mm Hg), O2 saturation (25.5 vs 94.1 %), and HCO3 (10.1 vs 23.4 mmol/L). On the other hand an increase in hemoglobin level (221.5 vs 120.3 g/L), haematocrit level (68.6 vs 45.2 %), and red blood cell count (10.4 vs 6.9 µL/1000) could be observed. CONCLUSION: our data clearly show that chronic systemic hypoxia causes massive ventricular hypertrophy accompanied by severe structural and morphological impairment of ventricular myocardium, which eventually results in cardiac failure.

Expression of hypoxia-inducible factor-1alpha (HIF-1alpha) related to oxidative stress in liver of rat-induced by systemic chronic normobaric hypoxia.

AIM: To observe the expression of hypoxia-inducible factor-1alpha (HIF-1alpha) and its relation with oxidative stress in liver of rats induced by systemic chronic normobaric hypoxia. METHODS: Twenty five male, 6-8 weeks old rats were induced by systemic hypoxia. Rats were divided randomly into 5 groups (n = 5 per group). The control group was exposed to normal environment while the hypoxic groups were kept in hypoxic chamber (10% O(2)) for 1, 3, 7, and 14 days. Animals were sacrificed, the liver isolated and homogenized. Total RNA was extracted and isolated and expression of HIF-1alpha mRNA was measured by real-time RT PCR using Pffafl method. Malondialdehyde (MDA), product of lipid peroxidation was measured by tBARS assay. Glutathione (GSH), an abundant endogenous antioxidant in the liver tissue was measured using Ellman method. RESULTS: Study showed that expression of HIF-1alpha mRNA was increased in group treated for 1 day of hypoxic condition, and then decreased in group treated for 3, 7 and 14 days of hypoxic condition related with duration of hypoxic condition. The MDA level in liver tissue increased, but not significant in all groups of hypoxic condition and persisted along duration time of hypoxic condition. The GSH level was decreased significantly (p<0.005) in all groups of hypoxic condition. CONCLUSION: Expression of HIF-1alpha mRNA was higher at the early phase of hypoxia and decreased as hypoxia continued. Systemic hypoxia induction caused increased ROS formation during hypoxia, and depleted the GSH concentration in the liver. Oxidative stress present in liver of rat was induced by systemic hypoxia.

Suppression of Rotenone-Treated Human Breast Cancer Stem Cell Survival Using Survivin Inhibitor YM155 is Associated to Oxidative Stress Modulation.

BACKGROUND: Despite recent progress in molecular-targeted therapies, breast cancer remains the primary leading cause of cancer related death among women worldwide. Breast cancer stem cells (BCSCs) are believed to be responsible for therapy resistance and cancer recurrence. We recently demonstrated that human BCSCs (CD24-/CD44+) could survive better than their counterpart non-BCSCs (CD24-/CD44-) when treated with rotenone, possibly due to lower levels of reactive oxygen species (ROS) production, high expression of antioxidant manganese superoxide dismutase (MnSOD), and anti-apoptotic survivin. The aim of this study was to verify the role of survivin on human BCSCs survival under oxidative stress modulation by suppressing its expression using YM155, a survivin inhibitor. METHODS: Human BCSCs (ALDH+ cells) were treated with YM155 for 24 h prior to treatment with rotenone for a further 6 h. We determined intracellular superoxide levels were determined using dihydroethidium assay, survivin and MnSOD expression using qRT-PCR, survivin protein level using ELISA, as well as cell viability using trypan blue exclusion and acridine orange/ethidium bromide apoptosis assay. RESULTS: Suppression of survivin expression using YM155 could reduce the survival of rotenone-treated BCSCs, which may be associated with oxidative stress modulation, as shown by increased ROS levels and decreased MnSOD expression. We confirm that survivin is responsible for maintaining BCSCs survival under oxidative stress modulation. Furthermore, YM155 could modulate oxidative stress in BCSCs by reducing MnSOD expression and increasing ROS levels. CONCLUSION: YM155 treatment could be used to overcome BCSCs resistance to oxidative stress-based anticancer therapies.

Chitosan exerts anticancer activity through induction of apoptosis and cell cycle arrest in oral cancer cells.

Chitosan, a multipurpose biomaterial, has been shown to exert effects against several types of cancer including oral cancer. However, the mechanisms underlying the anticancer activities of chitosan on oral squamous cell carcinoma (SCC) cells remain largely unknown. The present study aimed to compare the effects of low-molecular-weight chitosan (LMWC) and cisplatin on oral SCC Ca9-22 and non-cancer keratinocyte HaCaT cell lines. Cell viability and cell cycle profiles were measured by MTT assay and laser scanning cytometry, respectively. Apoptosis was examined by TUNEL assay and electron microscopy, followed by analysis of caspase activity. LMWC exhibited cytotoxic effects on Ca9-22, but not HaCaT cells, whereas cisplatin induced apoptosis in both types of cells. Exposure of Ca9-22 cells to LMWC led to G1/S cell cycle arrest and an increase of TUNEL-positive cells accompanied by an early apoptotic cell morphology and subtle increases of caspase activity. Short-term LMWC exposure was less cytotoxic to HaCaT cells than to Ca9-22 cells, and anticancer activity was exerted through induction of apoptosis and cell cycle arrest, suggesting that LMWC could be a promising natural anticancer agent with fewer side effects on normal cells.