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One of the most promising strategies in cancer therapy is to induce apoptotic pathway. For this purpose, several constructed agonists of Death Receptor 5 (DR5) are in clinical development. Extrinsic and intrinsic apoptosis pathways of various cancer cells are primarily induced through the activation of the proapoptotic DR5. The extracellular domain of DR5 is comprised of several functional domains, among them the cysteine-rich domains (CRDs) play a critical role in TRAIL-DR5-mediated apoptosis. It has recently been shown that the binding of agonistic monoclonal antibody to another N-terminal domain of DR5 could mediate its activation and apoptosis induction. Variable domains derived from heavy chain antibodies (hcAb) called VHHs or nanobodies are robust, efficient and smallest antigen binding fragments. These unique features of VHHs make them potential therapeutic and diagnostic candidates. In the present study, using phage display technology, a library containing VHH genes was generated of an immunized camel with hapten-peptide 1ITQQDLAPQQRA12 and used to isolate the binders of this peptide. Through screening the phage library, three binders with high binding ability to desired epitope in the NTR region were obtained. Considering to the key role of this epitope in apoptosis inducing, these selected binders could be potential candidates to trigger apoptosis in various cancer cells.

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Abstract- Silibinin a natural flavonoid has been reported to induce cell death in various types of cancers and also in endothelial cells which shows its anti-angiogenesis effect. However, its molecular mechanism is not clearly defined. In this article, we provided evidence for one of the mechanisms by which Silibinin induces apoptosis in HUVEC. For this purpose, HUVECs were grown on 96 well plates and cell viability was measured by MTT assay and IC50 was determined as 143μM after 24 hr of treatment by Silibinin. Caspase-9 activity in dose dependent (100-300μM) and time dependent (24,48 and 72hr) treatment by Silibinin was assessed using chromogenic substrate LEHD-pNA. Maximum activity of caspase 9 was in 100 μM of silibinin after 48 hours of treatment. DNA fragmentation was analyzed by gel electrophoresis. Cells were incubated with different concentrations of silibinin (100-400μM) and DNA that was extracted from cells which were incubated by 400 μM of silibinin formed a smear on agarose gel. Data obtained from this study showed the ability of Silibinin to inhibit HUVEC cell proliferation through apoptosis induction which indicates the anti-angiogenesis effect of this compound.

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Objective: Inhibition of apoptosis may favor the onset and progression of cancer. Survivin is an inhibitor of apoptosis that has been considered as a potential marker for diagnostic and/or prognostic of bladder cancer. The survivin protein regulates both cell division and cell death and is overexpressed in the vast majority of human cancers. In this study, the expression pattern and potential prognostic value of survivin was assessed in Formalin-Fixed Paraffin-Embedded (FFPE) samples of bladder tumor. Materials and Methods:FFPE samples, from patients with a well-known five-year survival record, were assessed by semi-quantitative RT-PCR technique. 51 samples from 30 patients were analyzed on the basis of Survivin expression. Tissue distribution and subcellular localization of survivin protein in tumor tissues was also examined by immunohistochemistry (IHC). Results: The expression of survivin was detected in 66.6% of the samples, with an increase of expression in higher grades of tumor. Furthermore, survivin was overexpressed in 2nd and 3rd recurrences of the same patients. Also, with the increased malignancy and accordingly increased expression of surviving, the overall 5-year survival rate of patients was significantly declined (P=0.036). IHC results also localized a nuclear localization for Survivin protein in tumor tissues. Conclusion: In conclusion, we were able to detect the expression of survivin in FFPE samples of bladder tissues, at the level of mRNA and protein and find a correlation between the level of Survivin expression and the degree of malignancy of the tumors. Our findings introduce Survivin as a suitable prognostic marker for predicting the bladder tumors.

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Objective: It is a firm belief that blood transfusion is life-saving in many situations, but at the same time transfusion complications could be life-threatening. The possible effects of blood Transfusion Related Immunomodulatory (TRIM) and its related mechanisms is one of the important debatable subjects in the field of blood transfusion medicine. One of the mechanisms through which transfusion can induced TRIM effects in recipient is apoptosis induction. Aim of this study was to investigate the apoptotic effects of stored blood in an in vitro model. Materials and Methods: To evaluate the apoptotic effects of blood storage, we studied the effect of the plasma (from whole blood) during storage on days 3, 10, 21 and 35 on Jurkat cells, which are sensitive to apoptosis .The plasma of whole blood was separated by centrifugation on different days. Then, Jurkat cells were cultured with plasma for 24 hours. Finally apoptosis level was studied by using flowcytometry for analyzing Annexin V on Jurkat cells (by SeroTec Annexin V:FITC Assay Kit). Results: The percentages of apoptosis on days 3, 10, 21 and 35 were 3.85±1.52, 5.27±2.12, 8.44±1.90, 12.01±2.32, respectively. The percentages of apoptotic cells in negative and positive control group was 3.85±1.94 and 65.80±2.28, respectively. Conclusion: The results of this study showed that plasma of whole blood have the apoptotic effects which enhanced during the storage of plasma. This in vitro model is also suitable for studying other TRIM related mechanisms.

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Background: Lung adenocarcinoma is the most primary histologic subtype of non-small cell lung cancer (NSCLC). Oxypeucedanin methanolate, a member of furanocoumarin, is a naturally occurring compound, which is isolated from Ferulago trifida Boiss, an endemic species in North-West of Iran.
Purpose: We attempt to uncover the capacities of oxypeucedanin methanolate to induce apoptosis and autophagy in NSCLC cells, as well as the underlying mechanism involved in this process.
Methods: The effect of oxypeucedanin methanolate on cell viability was evaluated on A549 cells by MTT assay. Flow cytometry assay was used to detect cellular apoptosis. Expression levels of BAX, caspase-3, BCL2 and LC3 in A549 cells were measured by Real time quantitative reverse transcription-polymerase chain reaction (Real time RT-PCR). A549 cells migration were analyzed using a wound‐healing assay.
Results: Oxypeucedanin methanolate inhibited A549 cell proliferation in dose- and time- dependent manner, as evaluated by MTT assay. The total apoptosis rate was (5.46%) for A549 cells not treated with oxypeucedanin methanolate. In contrast, the apoptosis rate was (29.6%) for A549 cells treated with oxypeucedanin methanolate at the concentration of 0.4 mM. Real time RT-PCR revealed that the mRNA expression of BAX, caspase-3 and LC3 were upregulated, while mRNA expression of BCL2 was downregulated. Untreated cell migration increased significantly after 72 hours.
Conclusion: Oxypeucedanin methanolate inhibits proliferation and it could induce apoptosis and autophagy of human non-small cell lung cancer cell line A549. Oxypeucedanin methanolate may be a good candidate for reducing of A549 cells metastasis. 

 

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Aims: Programmed cell death is a vital cellular process that is highly conserved in evolution. Apoptosis, as a common mode of programmed cell death, is disturbed in the most human malignancies and leads the resistance of cancers to current treatment strategies. Caspase 9 is a key protein in mitochondrial apoptosis. Activated Caspase 9 leads to activation of Caspase 3/7, initiating a caspase cascade and killing cell. In this study, Caspase 9 gene was cloned into pcDNA3.1(+) and its expression and function evaluated in cell.
Methods: PCR amplification of Caspase 9 was performed by specific primers and ligated into pcDNA3.1(+) after double-digestion with KpnI and BamHI. After sequencing, pcDNA/Caspase 9 was transfected into SH-SY5Y cells and treated with doxorubicin. Caspase 9 function was determined by its effect on cell death level by trypan blue and PI staining, and Caspase 3 activity, and its expression in cells measured by western blotting.
Finding: Caspase 9 gene cloning was done and its expression in cell defined by western blot. Overexpression of Caspase 9 led to autoprocessing following homodimerization and induction of cell death and also increased cell sensitivity to doxorubicin treatment and declined cell viability.
Conclusion: The cloned Caspase 9 was functional in cell and enhanced apoptosis in the treated cells by doxorubicin through self-activation and subsequently amplification of Caspase 3 activation.
 

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Objective: Curcumin, is the active component in turmeric (Curcuma long). This agent induces apoptosis via inhibiting various signaling pathways. However, its poor aqueous solubility prevents its widespread application. In this study, dendrosomes with water-solubility, nano-sized dimensions and nontoxic properties, was used for curcumin delivery to cells. Materials & Methods: In the present study, the potential of dendrosomes for use as a drug delivery system was assessed in AGS, HT3, 5637, hBMSC and U87 cell lines. In order to achieve optimal concentration of drug and the most suitable cell line, the effects of different concentrations of free and dendrosomal curcumin was examined on the cell lines. Propidium iodide staining was used for determining apoptosis induction and the expression of Bax gene was investigated by semi-Q RT-PCR. Results: Dendrosomal formulation significantly improved water solubility of highly hydrophobic curcumin in AGS cells. Flow cytometry analysis indicated 48 percent of cells treated with dendrosomal curcumin and 20 percent of cells treated with free curcumin (at the optimal concentration of drug) underwent apoptosis after 18h. Semi-Q RT-PCR results exhibited the increase of expression of Bax pro-apoptotic gene in cells treated with dendrosomal curcumin. Conclusion: Dendrosomal formulation, compared to free curcumin, enhanced curcumin solubility and increased apoptosis induction in treated cells. These data, together with the observation of a 50 % increase of Bax gene expression confirmed the apoptotic effects of dendrosomal formulation of curcumin.

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Objective: Cutaneous leishmaniasis is an endemic infectious disease considered to be a crucial health problem in many countries, including Iran. As such, there is a need for new medications with few side effects. In the present research we have studied the effect of artimisinin on  Leishmania major (L. major) and cell death in vitro. Methods: A specific number of promastigotes of L. major were grown in the presence of different concentration of artimisinin to achieve IC50 of the drug. The MTT method was applied to evaluate the cytotoxic effect of the artiminisinin on L. major. Various densities of this drug were applied to study the induction of apoptosis by flow cytometry on L. major promastigotes. Results: We calculated the IC50 of artimisinin to be 25 μg/mlby promastigote assay. Promastigotes were incubated at 72 hours incubation with various doses of artimisinin (10, 25, 50 and 100 μg/ml). The dose 100 μg/ml showed the most apoptosis (68.16%) by Annexin-V FITC. Whereas the 10 μg/ml dose had the least apoptosis (12.78%). There was no change in the control group. According to MTT, the toxic effect of artiminisinin on L. major promastigotes increased with increasing drug concentration. Conclusions: This study revealed that artimisinin has a little toxic effect on macrophages. According to the flow cytometry and MTT results, artimisinin can be suggested as an appropriate drug for in vivo antileishmanial study.

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Objective: Leishmaniasis is one of the significant causes of morbidity and mortality in several countries. It is an important problem in endemic areas such as Iran. The goal in treatment of leishmaniasis is to reduce the disease period and leave no evidence of any remaining scars or lesions. A derivative of artemisinin is artemether. Scientists believe that the strong action of artemether against parasites is due to the presence of an endoperoxide bridge. Due to problems in the treatment of Leishmania major, in this research we have studied the effect of artemether on Leishmania major under in vitro conditions. Methods: Parasites were cultured in NNN and RPMI, after which artemether at concentrations of 5, 10, 25, 50 and 100 μg/ml were used for the promastigote assay. Apoptosis was detected by flow cytometry and DNA ladder assay. Results: The inhibitory concentration (IC50) of artemether was determined to be 25 μg/ml. The percentage of apoptotic promastigotes at 25 μg/ml of artemether was 42.28. The results of DNA fragmentation show that exposure of Leishmania major promastigote cells to 25 μg/ml of artemether lead to DNA fragmentation. Conclusion: We have proven the effect of artemether on apoptosis of Leishmania major by flow cytometry and the DNA ladder assay.

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Objective: The goals of the study are evaluation the effect(s) of food deprivation as a social stress on testis structure. We also investigated the effects of melatonin treatment as an antioxidant component and inequality on the effect(s) of food deprivation. Methods: We investigated the improving effects of melatonin and social stress (food deprivation) on 42 male rats in 7 groups including control, sham, melatonin received (M), food deprivation (1/3 of control daily food) plus observation (FD), FD + melatonin (FDM), isolated FD (FDi), and FDi + melatonin (FDMi) groups. After 14 days, rats' testes were studied using immuno histochemistry and TUNEL assays to determine the number of apoptotic cells. Biochemical evaluation was taken on malodialdehide (MDA) and glutathione (GSH). ANOVA and Tukey's tests were done to analyse the data. PResults: The results of sham group was declined for similarity to results of control group. In FD group, MDA was increased significantly (PConclusion: Food deprivation can induce oxidative stress which is associated with increasment of apoptotic cells in testis. Isolation can compensate these effects. These results refer to inequality. Since melatonin is recognized for its anti-oxidative and improving effects, we have shown involvement of oxidative stress mechanisms on the stress of food deprivation with inequality.

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Objective: This study aimed to evaluate the incidence of apoptosis in vitrified and non-vitrified human ovarian tissue by the use of morphological analysis and apoptosis assay techniques. Methods: We obtained human ovarian tissue biopsies from 30 women who underwent elective caesarean sections. Tissues were transported to the laboratory in pre-warmed, equilibrated Leibovitz L-15 medium within 2 hours. The tissues were cut into small pieces and divided into two groups, vitrified and non-vitrified (control). Apoptosis incidence was assessed by light microscope and the TUNEL assay and DNA laddering. Evaluation of caspase 3/7 protein levels was performed by the luminescent assay. Results: We observed no morphological signs of apoptosis in the vitrified samples. There were no apoptosis signals as evidenced by TUNEL staining and no DNA laddering pattern observed in the vitrified group. Caspase 3/7 activity was 2294±169.19 RLU/µg protein in the non-vitrified control group and 2231±89.271 RLU/µg protein in the vitrified group, which was not significantly different. Conclusion: The structure of human vitrified ovarian tissue was well preserved. Vitrification could not increase apoptosis and caspase 3/7 activity in human ovarian tissue.

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Objective: Vitrification is a convenient, effective method for freezing and storing embryos. Under certain situations, such as an unsuitable endometrial environment, extra embryos can be re-vitrified for future use. There is inadequate data on the effects of re-vitrification on embryos, so we have evaluated the effects of re-vitrification on the development rate and expression of apoptotic and implantation genes. Methods: Female NMRI mice, ages six-eight weeks were super-ovulated with 7.5 IU PMSG and 7.5 IU hCG. Females were mated with males from the same strain and inspected for the presence of vaginal plugs the following morning. Females with the presence of vaginal plugs were considered to be pregnant and killed 62 h post hCG injection. Eight-cell embryos were flushed from their oviducts and subsequently divided into three experimental groups: fresh, vitrified-warmed 8-cell embryos, and re-vitrified-warmed blastocyst embryos. RNA was extracted and we used real-time PCR to evaluate expressions of Bax, Bcl-2, and ErbB4. Data was analyzed by the chi-square and ANOVA tests. Results: A significant difference existed in blastocyst formation rate, degeneration rate, and expressions of Bax, Bcl-2, and ErbB4 in re-vitrified embryos compared to fresh embryos. Conclusion: The vitrification and warming process did not affect the developmental rate and expressions of Bax, Bcl-2, and ErbB4 in the eight-cell stage embryos. However, we observed a change in development rate and expression rates of Bax, Bcl-2, and ErbB4 after re-vitrification in the early blastocyst stage.

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Abstract
Objective: Aging can affect adaption of the heart tissue apoptotic system to aerobic exercise and induction of doxorubicin. Therefore, the aim of this study was to evaluate the effect of pretreatment of aerobic training on doxorubicin-induced left ventricular apoptosis gene expression in aging model rats.
Methods: 42 adult Wistar male rats were randomly assigned to 6 groups of 7 rats: control of young, control of aging, aging + saline, aging + doxorubicin, aging + aerobic exercise + saline and aging + aerobic exercise + doxorubicin. Aging was induced by intraperitoneal injection of D- galactose (100mg/kg). The training protocol included treadmill running with a gradual increase between 25min/day to 54min/day and in velocity of 15m/min to 20m/min, 5 days/week for 6 weeks. During the two ultimate weeks of the training, animals underwent a 15 day intraperitoneal (i.p.) doxorubicin regimen, receiving 1 mg .kg−1 of doxorubicin per day. The rats were sacrificed 48 hours after the last training and injection session and part of the left ventricle was investigated by Real Time- PCR analysis to evaluate the expression of Bax and Bcl-2 genes expression.
Results: ANOVA revealed that doxorubicin injection induced a significant increase in expression of Bax and Bax/Bcl-2 ratio and an insignificant decrease in Bcl-2 gene expression. On the other hand, aerobic training before and during the induction of doxorubicin prevented the increase in Bax/Bcl-2 ratio and the doxorubicin-induced decrease in Bcl-2 gene expression.
Conclusion: Due to the significant decrease in Bax/Bcl-2 ratio in heart of the trained rats which were treated with doxorubicin, it can be concluded that aerobic exercise training prior to and during treatment with doxorubicin as a non-pharmacological strategy, probably protects cardiomyocytes from doxorubicin-induced apoptosis.

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Objective: Melanoma differentiation association gene-7 (MDA-7)/IL24 is a tumor suppressor gene. The iNGR peptide sequence, with excellent tropism to surface integrins has been employed for targeting of therapeutic molecules toward tumor cells. The purpose of our study was to construct a plasmid expressing modified MDA-7 fused with an iNGR peptide for better targeting to tumor cells. Methods: At first, we amplified the MDA-7 sequence by PCR, while the reverse primer contained the iNGR peptide sequence to add it to the end of a new MDA-7 gene. The resultant MDA7-iNGR and MDA-7 were cloned into a pCDNA3.1 eukaryotic expression vector. The accuracy of cloning methods, integrity of the plasmids, and sequence were sequentially evaluated by digestions, colony-PCR, and sequencing. The expressions of the plasmid constructs were assayed by ELISA following their transfection into Ad-293 cells. Next, the plasmids were transfected into Hep-G2 cells and their mRNA were converted to cDNA. We assessed the gene expression levels of Gadd153 and Bax. As the final step, apoptosis induction of Hep-G2 cells following transfection was evaluated by the help of PI/Annexin V staining according to flow cytometry. Results: The results showed the integrity of construct backbone in addition to reading frame of the MDA-7-iNGR sequence. A suitable expression/secretion of modified the MDA-7.iNGR protein was detected by ELISA assay of the culture supernatant when compared to the control construct that expressed unmodified MDA-7. The viabilty test demonstrated no benefit for this kind of modification of the MDA-7 protein. Real-time PCR and flow cytometry analyses revealed that the addition of iNGR to MDA-7 caused a decrease in its apoptotic effect on hepatic tumor cells compared to the normal protein. The modified protein had significant apoptosis induction compared to the negative control group (P<0.01). Conclusion:Although the new pCDNA/MDA-7.iNGR plasmid expressed iNGR-fused MDA-7 protein efficiently, it could not improve the natural apoptosis property of normal MDA-7.

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Objective: Despite many advances in cancer treatment and control, significant deficiencies remain and the rate of cancer growth is increasing. Due to the side effects of using chemicals and radiation, the use of herbs can have fewer side effects for the patient. The aim of this study was to investigate the potential anticancer effects of alcoholic extract of Lavender on inhibition and growth of HepG2 cell lines.
Materials and Methods: In this study, the effect of alcoholic extract of lavender (10,100,1000 and 1 μg / ml) on HepG2 liver cancer cell line was studied. MTT assay was used to evaluate the toxicity of lavender extract and cell viability. Cell cycle changes were assessed using a flow cytometer.
Oxygen-free species production and membrane lipid peroxidation were also measured.

Results: Results showed that alcoholic extract of Lavender has anti-cancer effect on HepG2 cell line. After confirming the effect of lavender extract on cell cycle, the results obtained from the evaluation of membrane lipid peroxidation and production of free reactive species were also significant.

Conclusion: According to the results of this study, the alcoholic extract of Lavender was reported as a potential anticancer agent and is one of the therapeutic strategies in liver cancer.