Combinatorial targeting of telomerase and DNA-PK induces synergistic apoptotic effects against Pre-B acute lymphoblastic leukemia cells.

BACKGROUND: Due to the high demand for novel approaches for leukemia-targeted therapy, this study investigates the impact of DNA-PK inhibitor NU7441 on the sensitivity of pre-B ALL cells to the telomerase inhibitor MST-312. METHODS: The study involved NALM-6 cells treated with MST-312 and NU7441, assessing their viability and metabolic activity using trypan blue and MTT assays. The study also evaluated apoptosis, gene expression changes, and DNA damage using flow cytometry, qRT-PCR, and micronucleus assays. The binding energy of MST-312 in the active site of telomerase was calculated using molecular docking. RESULTS: The study's findings revealed a synergistic decline in both cell viability and metabolic activity in NALM-6 cells when exposed to the combined treatment of MST-312 and NU7441, and this decrease occurred without any adverse effects on healthy PBMC cells. Furthermore, the combination treatment exhibited a significantly higher induction of apoptosis than treatment with MST-312 alone, as observed through flow cytometry assay. qRT-PCR analysis revealed that this enhanced apoptosis was associated with a notable downregulation of Bcl-2 expression and an upregulation of Bax gene expression. Moreover, the combination therapy decreased expression levels of hTERT and c-Myc genes. The micronucleus assay indicated that the combination treatment increased DNA damage in NALM-6 cells. Also, a good conformation between MST-312 and the active site of telomerase was revealed by docking data. CONCLUSIONS: The study suggests that simultaneous inhibition of telomerase and DNA-PK in pre-B ALL presents a novel targeted therapy approach.

Topical ocular administration using thermosensitive chitosan-glycerophosphate-PRP hydrogels for improved ocular bioavailability.

PURPOSE: One of the difficulties in the pharmacy field is the delivery of drugs for the eyes. Topical therapy is one of the most common methods for treating eye diseases. Due to their unique properties, including biocompatibility and suitable degradation, hydrogels are appropriate for biological purposes. Platelet-rich plasma (PRP), as a designated concentration of platelets, is in a smaller volume than the plasma and is considered a rich source of growth factor that has been used in recent years, including applications in eye diseases including corneal wound healing, improvement of dry eye and post-LASIK syndrome. METHODS: The present study was performed to fabricate Chitosan (CS) and glycerophosphate (GP) based hydrogels that are temperature-sensitive for PRP and investigate their effect on ocular stem cells. RESULTS: CS-GP-based temperature-sensitive hydrogels containing PRP were successfully fabricated using CS and GP. This hydrogel is liquid at ambient temperature and a gel at ocular temperature. Rheology, FTIR, and SEM tests assessed the properties of the hydrogels. The results of the MTT test showed that the hydrogel made with the optimal formulation was not toxic to LSC cell lines. CONCLUSIONS: Given this, CS-GP-based hydrogels can be applied as a biocompatible formulation in ocular medication administration with increased bioavailability at the ocular surface and topical delivery of PRP.

Association of IKZF1 and CDKN2A gene polymorphisms with childhood acute lymphoblastic leukemia: a high-resolution melting analysis.

BACKGROUND: Acute lymphoblastic leukemia is the most prevailing pediatric hematologic malignancy, and various factors such as environmental exposures and genetic variation affect ALL susceptibility and patients outcome. According to genome-wide association studies, several single nucleotide polymorphisms (SNPs) in IKZF1 (rs4132601) and CDKN2A (rs3731249 and rs3731217) genes are associated with ALL susceptibility. Hereupon, this study aimed to discover the association between these SNPs and the risk of childhood ALL among a sample of the Iranian population. METHODS: A total of fifty children with ALL were included in this case-control study, along with an additional fifty healthy children, matched for age and gender. High-resolution melting (HRM) analysis was employed to genotyping rs4132601, rs3731249, and rs3731217. RESULTS: In the patient group, the CT genotype and T allele frequency of rs3731249 were significantly greater than controls (p = 0.01 and p = 0.005, respectively). Moreover, the positive association of CT and dominant model (CT + TT) genotypes and T allele at rs3731249 with the risk of ALL was confirmed (OR = 9.56, OR = 10.76 and OR = 11.00, respectively). There was no significant relation between rs4132601 (IKZF1), rs3731217 (CDKN2A), and childhood ALL. CONCLUSION: The present study indicates that CT genotype and T allele at rs3731249 (CDKN2A) can significantly increase the risk of ALL among children.

Promising anti-leukemic effect of Zataria multiflora extract in combination with doxorubicin to combat acute lymphoblastic leukemia cells (Nalm-6) (in vitro and in silico).

One of the heterogeneous hematologic malignancies of the lymphocyte precursors is ALL. ALL has two incidence peaks that were determined in 2-5 years children and 60 years old adults. Cardiotoxicity of chemotherapeutic drugs is one of important side effects which may occur during or after chemotherapy period. The aim of this study was to evaluate the effect of ZME, Dox, and combinations on Nalm-6 cells. In this vein, the cell viability was assessed by Trypan blue and MTT assay. Evaluation of apoptosis was also analyzed by Annexin-V/PI staining. Moreover, the expression of Bax, Bcl-2, Bcl-xl, hTERT, c-Myc, P53, and P21 genes was detected by Real-Time PCR. Molecular docking as an in-silico method was performed for Bcl-2 and Bcl-xl proteins as well. Our achievements indicated that ZME had dose-dependent effect on Nalm-6 cells and ZME synergistically potentiated Dox effect. The expression of Bax, P53 and P21 genes increased although the expression of Bcl-2 genes decreased when cells treated with ZME/ Dox combination. Molecular docking showed the interactions of carvacrol and thymol in the active cavities of BCL2 and BCL-xl. Regarding to present study, ZME could be utilized as a combinatorial and potential drug for leukemic patients, which is under the treatment by Dox due to reducing the chemotherapy drug doses.

Association of DNA repair genes polymorphisms with childhood acute lymphoblastic leukemia: a high-resolution melting analysis.

OBJECTIVE: Acute lymphoblastic leukemia (ALL) is one of the most common cancers in children for which the exact pathogenesis is not yet known. Single-nucleotide variants (SNVs) in different DNA repair genes are reported to be associated with ALL risk. This study aimed to determine the association between XRCC1 (rs1799782) and NBN (rs1805794, rs709816) SNVs and childhood ALL risk in a sample of the Iranian population. Fifty children with ALL and 50 age- and sex-matched healthy children were included in this case-control study. Genotyping of the mentioned SNVs was done by high-resolution melting (HRM) analysis. RESULTS: The prevalence of all three SNVs in XRCC1 and NBN genes did not differ between the patient and control groups, and these polymorphisms were not associated with childhood ALL risk (P > 0.05). HRM was a practical method for the detection of SNVs in XRCC1 and NBN genes. We found no significant association between XRCC1 (rs1799782) and NBN (rs1805794, rs709816) SNVs and childhood ALL risk.

Platelet Microparticles Accelerate Proliferation and Growth of Mesenchymal Stem Cells through Longevity-Related Genes.

BACKGROUND: Due to their self-renewal and differentiation ability, the mesenchymal stem cells (MSCs) have been studied extensively. However, the MSCs lifespan is restricted; they undergo several divisions in vitro that cause several alternations in cellular features and relatively lessens their application. Thus, this study was aimed to assess the effect of platelet-derived microparticles (PMPs), a valuable source of proteins, microRNAs (miRNAs), and growth factors, on the expression of hTERT, c-MYC, p16, p53, and p21 as the most important aging and cell longevity genes alongside with population doubling time (PDT) of PMP-treated cells in comparison to a control group. METHODS: Umbilical cord MSCs (UC-MSCs) were used in this study, whereby they reached a confluency of 30%. MSCs were treated by PMPs (50 µg/mL), and then, PDT was determined for both groups. Quantitative expression of hTERT, c-MYC, p16, p53, and p21 was examined through quantitative real-time PCR at various intervals (i.e. after five and thirty days as well as freezing-thawing process). RESULTS: Our results demonstrated that the treated group had a shorter PDT in comparison to the control group (P<0.050). The real-Time PCR data also indicated that PMPs were able to remarkably up-regulate hTERT and c-MYC genes expression while down-regulating the expression of p16, p21, and p53 genes (P<0.050), especially following five days of treatment. CONCLUSION: According to these data, it appears that PMPs are a safe and effective candidate for prolonging the lifespan of UC-MSCs; however, further investigations are needed to corroborate this finding.

Inhibition of DNA damage response pathway using combination of DDR pathway inhibitors and radiation in treatment of acute lymphoblastic leukemia cells.

An alarming increase in acute lymphoblastic leukemia cases among children and adults has attracted the attention of researchers to discover new therapeutic strategies with a better prognosis. In cancer cells, the DNA damage response (DDR) pathway elements have been recognized to protect tumor cells from various stresses and cause tumor progression; targeting these DDR members is an attractive strategy for treatment of cancers. The inhibition of the DDR pathway in cancer cells for the treatment of cancers has recently been introduced. Hence, effective treatment strategies are needed for this purpose. Chemotherapy in combination with radiotherapy is considered a potential therapeutic strategy for acute leukemia. This review aims to assess the synergistic effects of these inhibitors with irradiation for the treatment of leukemia.

Lay abstract Acute lymphoblastic leukemia (ALL) is a blood malignancy that is caused by the high proliferation of lymphoid precursors in bone marrow and peripheral blood, and it is one of the common malignancies among children and adults. Novel therapeutic strategies are increasingly being studied to suppress cancer cells. DNA damage response pathways have developed in cancer cells that protect tumor cells from chemotherapy and radiotherapy and cause relapse and treatment failure. Effective drugs in ALL treatment can suppress cancer cells through several mechanisms, such as inducing DNA damage in cancer cells. However, due to the active repair systems in cancer cells, treatment may fail. Recently, the inhibition of these repair pathways and the combination of DNA damage response pathway inhibitors with chemotherapy and radiotherapy in cancer treatment have been studied. The purpose of this review is to outline the synergistic effects of DNA damage response pathway inhibitors and radiation in the death of leukemic cells.

High intensity interval training improves diabetic cardiomyopathy via miR-1 dependent suppression of cardiomyocyte apoptosis in diabetic rats.

PURPOSE: Diabetes and its complications such as diabetic cardiomyopathy still account for significant morbidity and mortality. High-quality evidence was shown the importance of exercise in controlling diabetes complications, but the molecular mechanism on diabetic cardiomyopathy is not yet fully understood. This study aimed to compare and investigate the effect of high intensity interval training (HIIT) and continuous endurance training (CET) on the signaling pathway of diabetic cardiomyopathy. METHODS: Hence, 21 Wistar rats with an average weight of 260 ± 10 g, after induction of diabetes (STZ 50 mg/kg BW) were randomly divided into three groups (control, CET and HIIT; n = 7). Training programs were conducted 5 days a week for 5 weeks. CET program was defined as running at 60% vVO2max for 30 min in each session and the HIIT program was defined as running at 85-90% vVO2max for 3 min followed by 1 min recovery (30-35% vVO2max), that was repeated four times in each session. The cardiac performance was analyzed via determination of end systolic and diastolic dimensions and the ejection fraction by echocardiography. To elucidate the responsible molecular mechanism of miR-1, IGF-1 and IGF-1R mRNA and apoptosis marker protein expression were investigated. RESULTS: Both training programs specifically HIIT, significantly reduced the blood glucose, enhanced heart performance, reduced miR-1 expression, induced IGF-1 and IGF-1R expression and reduced apoptotic protein expression. CONCLUSION: We showed that HIIT is more effective than CET for reduction of diabetic cardiomyopathy as a complication of diabetes in animal models through suppressing miR-1 and its downstream apoptosis pathway.

Gene expression analysis of activating and inhibitory receptors of natural killer cells in patients with acute myeloblastic leukemia.

PURPOSE: Natural killer (NK) cells are cytotoxic lymphocytes, which have long been known to play an essential role in immune surveillance of tumor cells. The results of several clinical studies imply evidence of impaired activity of NK cells in acute myeloblastic leukemia (AML). The aim of this study was to investigate the gene expression of activating and inhibitory receptors of NK cells in patients with newly diagnosed AML before and after induction therapy using 7 + 3 regimen in comparison to healthy donors. MATERIALS AND METHODS: Sixteen AML patients aged 16-64 years as well as 16 matched healthy individuals were studied. Peripheral blood samples from patients were obtained in two steps, namely, in newly diagnosed patients and 28 days after receiving induction therapy. Real-time PCR was performed to evaluate the expression levels of activating receptors, including DNAM-1 and NKp46 as well as inhibitory receptors of KIR2DL1 and NKG2A. RESULTS: Our results demonstrated that the newly diagnosed patients showed over 50% decrease in NKp46 expression and a 6-fold increase in KIR2DL1 expression compared to healthy controls. The mRNA expression analysis in patients after induction therapy suggested a significant decrease in mRNA expressions of KIR2DL1 and NKG2A in comparison to newly diagnosed patients. CONCLUSION: Herewith, we show a statistical difference in mRNA expression levels of activating (NKp46) and inhibitory receptors from NK cells in newly diagnosed AML patients when compared with healthy controls or patients who received induction therapy, supporting the findings of researchers who reported the impaired NK cells cytotoxicity in AML patients.

Differential regulatory effects of chemotherapeutic protocol on CCL3_CCL4_CCL5/CCR5 axes in acute myeloid leukemia patients with monocytic lineage.

AIMS: AML (Acute myeloid leukemia) is characterized as a heterogeneous cancer. Chemokines play fundamental roles in the onset, progression cellular, migration, survival and improvement of AML therapy outcomes. The CCR5 receptors together with their ligands have indirect effects on the progression of cancer. In the present study, we have decided to investigate the impact of chemotherapy on the expression of CCR5 and its related ligands (CCL5, CCL4 and CCL3). MAIN METHODS: In this study, peripheral blood and bone marrow specimens were collected prior and post the first stage of (7 + 3) chemotherapy from 25 AML-M4/M5 patients. The expression of CCR by Lymphocytes in peripheral blood was examined by flow cytometry and QRT-PCR. The serum levels of chemokines were measured by ELISA. KEY FINDINGS: There was not observed leukemic blast cells in peripheral blood smear at post first stage of chemotherapy. We found that the expression of CCR5 was attenuated in patients post the first stage of chemotherapy and the healthy control subjects. We have also observed that the serum levels of chemokines were elevated in AML patients prior to chemotherapy. Although in post-chemotherapy stage, only CCL3 was found to reach to the baseline level, CCL5 and CCL4 have not returned to the basal level and were significantly higher than healthy control subjects. SIGNIFICANCE: The current chemotherapy protocol was not able to completely inhibit CCL5 and CCL4. In conclusion, our findings in harmony with previous studies suggest that inhibition of chemokines along with chemotherapy in AML patients may aid therapy.

Analysis of the reannealing- instead of melting-curve in the detection of JAK2 V617F mutation by HRM method.

BACKGROUND: The Janus kinase 2 (JAK2) has an important role in the intracellular signaling in normal and neoplastic cells. JAK2 mutation, called JAK2 V617F, is frequently found in Philadelphia chromosome-negative myeloproliferative neoplasms. We aimed to assess the analytical efficiency of high-resolution melting (HRM) method using reannealing-curve analysis in comparison with routine melting-curve analysis for JAK2 V617F mutation detection. METHOD: Twenty-three samples including one negative synthetic standard DNA, two 50% and 75% positive synthetic standard DNA samples, five wild-type samples and 15 samples positive for JAK2 V617F were examined by HRM. Melting and reannealing stages were performed, and then, raw and normalized curves were compared between the two stages. RESULTS: In melting-curve analysis, the wild-type and mutant samples had different melting temperatures (75/53°C and 75/10°C, respectively). In normalized curves corresponding to reannealing method, mutant samples were better separated from the baseline than in melting method as well as for samples with different mutant DNA burden from each other. Furthermore, wild-type samples were more homogenous in the normalized curves corresponding to reannealing than in melting method. This means that patients with a low allelic burden may be wrongly interpreted as normal in the common melting method. CONCLUSION: We suggest the use of reannealing instead of the melting-curve analysis for the detection of sequence variations, especially for large-scale mutation and allele burden measurement in clinical settings. However, more evaluations with more sample size will better improve the benefits of reannealing-curve analysis in research and clinic.

Telomerase inhibitor MST-312 induces apoptosis of multiple myeloma cells and down-regulation of anti-apoptotic, proliferative and inflammatory genes.

AIMS: The telomerase-based therapy of cancer has received a great deal of attention due to the fact that it is expressed in almost all of the cancer cells while it is inactivated in most of the normal somatic cells. Current investigation was aimed to examine the effects of namely telomerase inhibitor, the MST-312, as a chemically modified derivative of epigallocatechin gallate (EGCG), on human multiple myeloma cell line U-266. MAIN METHODS: U-266 cells were cultured and then treated by MST-312. The viability of cultured cells was measured by both trypan blue staining and MTT assay techniques. To examine the apoptosis, annexin-V/7-AAD staining using flow cytometry method was employed. To analysis the expression of Bax, Bcl-2, c-Myc, hTERT, IL-6 and TNF-α genes, the quantitative real-time PCR was employed. KEY FINDINGS: We observed the short-term dose-dependent cytotoxic and apoptotic effect of MST-312 against U-266 myeloma cells. Gene expression analysis indicated that the MST-312-based apoptosis was associated with up-regulation of pro-apoptotic gene (Bax) as well as down-regulation of anti-apoptotic (Bcl-2), proliferative (c-Myc, hTERT) and inflammatory (IL-6, TNF-α) genes. SIGNIFICANCE: These findings suggest that telomerase-based therapy using MST-312 may represent a novel promising strategy for treatment of multiple myeloma.

A fibrinous and allogeneic fibroblast-enriched membrane as a biocompatible material can improve diabetic wound healing.

The application of conventional approaches to diabetic wound regeneration has some limitations. Thus, skin substitutes could be a new therapeutic possibility. In this regard, fibrin scaffolds are promising materials due to their desirable characteristics. Since defective fibroblasts caused by diabetes can disrupt regeneration, it seems that the use of living cells can improve the healing process. Thus, based on this fact, a cellular fibrin membrane was used to evaluate the diabetic wound healing in rats. The fibrin membrane was fabricated using fresh frozen plasma on which isolated fibroblasts were cultured. The wound model was created on 36 diabetic rats that were randomly divided into three groups: control, membrane, and cellular fibrin membrane (CM). Wound photogramography and immuno-histopathological staining were performed during consecutive days after treatment. Macroscopic evaluation of the wounds indicated a noteworthy enhancement of wound closure in the CM group. In the CM group, the re-epithelialization rate on day 7, 10 (p < 0.001), and 14 (p < 0.05), the fibroblast percentage on day 3 (p < 0.01) and 7 (p < 0.05) and the collagenization in all days were significantly higher than those of other groups (p < 0.001). The fibroblast number in the CM group on day 10 was significantly (p < 0.01) lower than that in the other groups. Contrary to the neutrophil and angiogenesis percentages that had no significant difference among the groups at different points of time (p > 0.05), the macrophage percentage on day 7 (P < 0.01), 10, and 14 (p < 0.05) was significantly lower in the CM group as compared to that in other groups. Overall, it seems that the use of a fibroblast-loaded fibrin membrane is an attractive strategy to promote diabetic wound healing.

The telomerase inhibitor MST-312 synergistically enhances the apoptotic effect of doxorubicin in pre-B acute lymphoblastic leukemia cells.

BACKGROUND: There have been consistent efforts in concomitant administration of chemotherapeutic agents such as doxorubicin beside other more tumor-specific drugs in order to increase sensitivity of tumor cells to the cytotoxicity of doxorubicin and reduce necessary chemotherapeutic dosage. Telomerase-targeted therapy for cancer has received great attention because telomerase is expressed in almost all cancer cells but is inactive in most normal somatic cells. The present study was aimed to investigate the effect of telomerase inhibitor MST-312, a chemically modified derivative of epigallocatechin gallate (EGCG), on doxorubicin-induced apoptosis in pre-B acute lymphoblastic leukemia cells. MATERIALS AND METHODS: The pre-B ALL cell lines (NALM-6 and REH) were cultured and treated with MST-312 and doxorubicin, separately and in combination. Cell viability was measured by trypan blue staining and MTT assay. Annexin-V/7-AAD staining by flow cytometry was used for evaluation of apoptosis. Gene expression of hTERT, c-Myc, Bax and Bcl-2, was detected by the quantitative Real-Time PCR. RESULTS: Our results showed that MST-312 exerted dose-dependent short-term cytotoxic and apoptotic effects on pre-B ALL cells. Combination of MST-312 and doxorubicin synergistically enhanced the cytotoxicity and apoptosis of both NALM-6 and REH cells. Consistently, apoptosis induction by the combined treatment was associated with significant reduction of Bcl-2, c-Myc and hTERT, and a marked increase of Bax mRNA expression levels, compared with the single treatments. CONCLUSIONS: Our data suggest that the synergistic anticancer effect induced by combination of MST-312 and doxorubicin represents a novel treatment strategy for pre-B ALL.

In vitro evaluation of decontamination effects on mechanical properties of fibrin membrane.

Background: Tissue engineering has been investigated as a potential method for healing traumatized tissues. Biomaterials are material devices or implants used to repair or replace native body tissues and organs. The present study was conducted to evaluate the effects of decontamination methods on biological/mechanical properties and degradation/adhesion test of the platelet-rich fibrin (PRF) membranes to compare these properties with intact membranes as a biological biomaterial. Methods: The in vitro degradation tests were conducted by placing the equal sizes of (i) intact PRF membrane, (ii) PRF membrane sterilized by autoclave (iii), ultraviolet (UV), and (iiii) gamma irradiation in phosphate buffer solution on a shaker. The degradation profiles were expressed. Adhesion test was performed by counting adhered mouse fibroblast and sterilized fibrin membrane was compared to normal fibrin membrane by different sterilization methods. Results: The preliminary findings of sterilized PRF membranes showed that UV exposure (p<0.05) and autoclaved fibrin membranes (p<0.01) have significantly lower degradability compared to normal fibrin membranes. Gamma irradiation is similar to normal membrane in degradability. Cell adherence in all groups of fibrin membrane was significantly lower than the group without membrane, but there was no significant difference between intact and sterilized groups of fibrin membranes. Conclusion: Sterilization of fibrin membrane with different protocols does not have any adverse effects on cell adhesion; however, cell adherence is naturally very weak even in normal membranes. Also, it seems that ultraviolet ray polymerizes fibrin filaments and merges them to each other and increases the ability of fibrin membrane against degradation. Autoclaved fibrin membrane content proteins are denatured because of pressure and heat and show an increase in hardness and stability against degradation.

A New Indole Derivative Decreased SALL4 Gene Expression in Acute Promyelocytic Leukemia Cell Line (NB4)

Background: Acute myeloblastic leukemia (AML) is a clonal disorder due to bone marrow failure and uncontrolled proliferation of myeloid lineage. Acute promyelocytic leukemia (APL) is a subtype of AML. Heterocyclic compounds, such as indole, are considered as attractive candidates for cancer therapy, due to their abundance in nature and known biological activity. Sal-like protein (SALL4) is a zinc finger transcription factor involving in the multi-potency of stem cells, in the NB4 cell line. This study was aimed to evaluate the effects of basal indole and its new derivative, 2-(1-((2, 4-Aril)imino)-2,2,2-trifluoroethyl) phenyl-1H Indole-3- carbaldehyde (TFPHC), on the expression of SALL4. Methods: Cells were cultured and treated with different concentrations (75, 150, and 300 µg/mL) of the new indole derivative and DMSO, as a vehicle control, for 24 and 48 hours. Cell proliferation was evaluated by using Trypan blue exclusion and MTT assays. The percentage of apoptotic cells was determined by flowcytometry analysis using the Annexin V/PI apoptosis detection kit; mRNA expression of SALL4 was studied using absolute quantitative RT-PCR. Data were analyzed by student's t-test. P<0.05 were considered statistically significant. Results: Our findings demonstrated the effects of new indole derivatives on SALL4 mRNA expression. Expression of SALL4 mRNA was significantly decreased at 75, 150, and 300 µg/mL concentrations. Conclusion: SALL4 plays a role in the survival of APL cells. SALL4 expression could be suppressed by the novel indole derivative. Additionally, SALL4 gene suppression can serve as a target in APL therapy.

Elevation of cAMP Levels Inhibits Doxorubicin-Induced Apoptosis in Pre- B ALL NALM- 6 Cells Through Induction of BAD Phosphorylation and Inhibition of P53 Accumulation.

Recognition of the molecular mechanisms of cAMP action against DNA damage-induced apoptosis can be useful to improve the efficacy of DNA damaging therapeutic agents. Considering the critical role of bcl-2-associated death promoter (BAD) and p53 proteins in DNA damage -induced apoptosis, the aim of this study was to assess the effect of cAMP-elevating agents on these proteins in doxorubicin-treated pre-B acute lymphoblastic leukemia (pre-B ALL) NALM-6 cells.The pre-B ALL cell line NALM-6 was cultured and treated with doxorubicin in combination with or without cAMP-elevating agents forskolin and 3-isobutyl-1-methylxanthine (IBMX). Cell viability was measured by trypan blue staining and MTT assay. For evaluation of apoptosis, annexin-V staining by flow cytometry and caspase-3 activity assay were used. Protein expression of p53, BAD and phoshorylated BAD was detected by western blotting analysis.cAMP-increasing agents diminished the doxorubicin-mediated cytotoxicity in NALM-6 cells as indicated by the viability assays. Annexin-V apoptosis assay showed that the cAMP-elevating agents decreased doxorubicin-induced apoptosis. Moreover, doxorubicin-induced caspase-3 activity was attenuated in the presence of cAMP-increasing agents. Western blot results revealed the reduced expression of p53 protein in cells treated with combination of cAMP-elevating agents and doxorubicin in contrast to cells treated with doxorubicin alone. Expression of total BAD protein was not affected by doxorubicin and cAMP-elevating agents. However, phosphorylation of BAD protein was induced in the presence of cAMP-elevating agents. Our study suggests that elevated cAMP levels inhibit doxorubicin-induced apoptosis in pre-B ALL cells through induction of BAD phosphorylation and abrogation of p53 accumulation.

MST-312 induces G2/M cell cycle arrest and apoptosis in APL cells through inhibition of telomerase activity and suppression of NF-κB pathway.

Telomerase-targeted therapy for cancer has received great attention because telomerase is expressed in almost all cancer cells but is inactive in most normal somatic cells. This study was aimed to investigate the effects of telomerase inhibitor MST-312, a chemically modified derivative of epigallocatechin gallate (EGCG), on acute promyelocytic leukemia (APL) cells. Our results showed that MST-312 exerted a dose-dependent short-term cytotoxic effect on APL cells, with G2/M cell cycle arrest. Moreover, MST-312 induced apoptosis of APL cells in caspase-mediated manner. Telomeric repeat amplification protocol (TRAP) assay revealed significant reduction in telomerase activity of APL cells following short-term exposure to MST-312. Interestingly, MST-312-induced telomerase inhibition was coupled with suppression of NF-κB activity as evidenced by inhibition of IκBα phosphorylation and its degradation and decreased NF-κB DNA binding activity. In addition, gene expression analysis showed downregulation of genes regulated by NF-κB, such as antiapoptotic (survivin, Bcl-2, Mcl-1), proliferative (c-Myc), and telomerase-related (hTERT) genes. Importantly, MST-312 did not show any apoptotic effect in normal human peripheral blood mononuclear cells (PBMCs). In conclusion, our data suggest that dual inhibition of telomerase activity and NF-κB pathway by MST-312 represents a novel treatment strategy for APL.

Genetic Variants of Cytochrome b-245, Alpha Polypeptide Gene and Premature Acute Myocardial Infarction Risk in an Iranian Population.

BACKGROUND: Oxidative stress induced by superoxide anion plays critical roles in the pathogenesis of coronary artery disease (CAD) and hence acute myocardial infarction (AMI). The major source of superoxide production in vascular smooth muscle and endothelial cells is the NADPH oxidase complex. An essential component of this complex is p22phox, that is encoded by the cytochrome b-245, alpha polypeptide (CYBA) gene. The aim of this study was to investigate the association of CYBA variants (rs1049255 and rs4673) and premature acute myocardial infarction risk in an Iranian population. METHODS: The study population consisted of 158 patients under the age of 50 years, with a diagnosis of premature AMI, and 168 age-matched controls with normal coronary angiograms. Genotyping of the polymorphisms was performed by the polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP). RESULTS: There was no association between the genotypes and allele frequencies of rs4673 polymorphism and premature acute myocardial infarction (P>0.05). A significant statistical association was observed between the genotypes distribution of rs1049255 polymorphism and AMI risk (P=0.037). Furthermore, the distribution of AA+AG/GG genotypes was found to be statistically significant between the two groups (P=0.011). CONCLUSIONS: Our findings indicated that rs1049255 but not rs4673 polymorphism is associated with premature AMI.

Association of platelet collagen receptor polymorphisms with premature acute myocardial infarction.

The impact of platelet collagen receptor polymorphisms in the pathogenesis of myocardial infarction at young age remains unknown. To determine whether either of the two platelet collagen receptor polymorphisms (GP VI T13254C and GP Ia C807T) was associated with premature acute myocardial infarction. One hundred patients with premature acute myocardial infarction and 100 age-matched controls with normal coronary angiograms were studied. Genotyping was done using PCR followed by restriction fragment length polymorphism (RFLP). GP Ia C807T polymorphism was more frequent in the patient group (65%) than in the control group (53%). However, there was no association between this polymorphism and premature acute myocardial infarction (P = 0.08). The prevalence of T13254C polymorphism did not differ between patients (38%) and controls (33%), and this polymorphism was not associated with premature acute myocardial infarction (P = 0.46). Logistic regression analysis also indicated no association between these polymorphisms and premature acute myocardial infarction (C807T with P = 0.51 and T13254C with P = 0.20). There is no association between GP VI T13254C or GP Ia C807T polymorphisms and premature acute myocardial infarction.