Formononetin and Dihydroartemisinin Act Synergistically to Induce Apoptosis in Human Acute Myeloid Leukemia Cell Lines.

OBJECTIVE: Enhanced cell survival and drug resistance in tumor cells have been linked to the overexpression of antiapoptotic members of the Bcl-2 family proteins, including Bcl-2 and Mcl-1. The aim of this study was to explore the impact of formononetin and dihydroartemisinin combination on the growth and apoptosis of acute myeloid leukemia (AML) cells. MATERIALS AND METHODS: In this experimental study, the cell survival and cell proliferation were tested by MTT assay and trypan blue staining. The evaluation of cell apoptosis was conducted using Hoechst 33342 staining and a colorimetric assay to measure caspase-3 activity. To determine the mRNA levels of Mcl-1, Bcl-2, Bax, and Cyclin D1, a quantitative real-time polymerase chain reaction (qRT-PCR) was performed. RESULTS: We showed that treatment with either formononetin or dihydroartemisinin alone, led to significant decrease in the cell survival and growth, and triggered apoptosis in U937 and KG-1 AML cell lines. Moreover, treatment with each of the compounds alone significantly decreased the mRNA levels of Mcl-1, Bcl-2 and Cyclin D1 mRNA, while, the expression level of Bax mRNA was enhanced. Combination of two compounds showed a synergistic anti-cancer effect. CONCLUSION: The anti-leukemic potential of formononetin and dihydroartemisinin is exerted through the effect on cell cycle progression and intrinsic pathway of apoptosis. Therefore, they can be considered as a potential anti-leukemic agent alone or along with existing chemotherapeutic drugs.

The Combination of 5-FU and Resveratrol Can Suppress the Growth of Glioblastoma Cells Through Downregulation of TRPM2 and ß-Catenin.

Glioblastoma multiforme (GBM) is the most common as well as the most fatal primary malignant tumor of the central nervous system (CNS), which still lacks a definitive cure. 5-FU is an anti-metabolite anti-cancer agent which has shown promising results for GBM treatment. Resveratrol (Res) is a phytochemical anti-oxidant that has also been effective in suppressing the progression of GBM. The combination of 5-FU and Res has been studied in a variety of cancers, but no study has assessed this combination in GBM. In this study, we investigated how 5-FU and Res, in combination and alone, may affect the growth and apoptosis of GBM cells and also the potential of TRPM2 and ß-catenin as the mediator of their effects. U87 cells were cultured as the in vitro model. MTT assay was used for measuring cellular growth, and RT-qPCR was used to measure the level of caspase-3, TRPM2, and ß-catenin; caspase-3 level served as the indicator of apoptotic rate. 5-FU and Res, in combination and alone, suppressed the growth while promoting the apoptosis of U87 cells; these effects were significantly greater when they were used in combination. RT-qPCR showed downregulation of TRPM-2 and ß-catenin in response to this combination, which suggested that these two molecules may mediate the cited anti-oncogenic effects. In conclusion, our study confirmed the synergism between 5-FU and Res in suppressing the progression of GBM and suggested the putative axis of TRPM2/ ß-catenin as the downstream mediator of this therapeutic regime. Future studies may be able to approve the eligibility of this therapeutic regime for GBM treatment and also the underlying mechanism.

Dihydroartemisinin Enhances the Therapeutic Efficacy of BH3 Mimetic Inhibitor in Acute Lymphoblastic Leukemia Cells via Inhibition of Mcl-1.

INTRODUCTION: Up-regulation of the anti-apoptotic proteins such as Mcl-1 is associated with the primary and secondary resistance of tumor cells to ABT-737 Bcl-2 inhibitor. The combined treatment of Bcl-2 inhibitors with Mcl-1 inhibitors has been proposed as an attractive therapeutic strategy to overcome this drug resistance. Here, we investigated the effect of dihydroartemisinin on Mcl-1 expression and sensitization of T-ALL cells to ABT-737. METHODS: The cell growth and survival were tested by the cell proliferation and MTT assays, respectively. The mRNA levels of Bcl-2, Mcl-1, Bax and P21 were examined by qRT-PCR. Apoptosis were detected by Hoechst 33342 staining and caspase-3 activity assay. RESULTS: Our data showed that combination treatment with dihydroartemisinin and ABT-737 caused a significant decrease in the IC50 value and synergistically reduced the cell survival compared with dihydroartemisinin or ABT-737 alone. ABT-737 enhanced the Mcl-1 mRNA expression. Dihydroartemisinin also down-regulated the expression of Bcl-2 and Mcl-1 and enhanced the P21 and Bax expression. Moreover, dihydroartemisinin enhanced the apoptosis induced by ABT-737 in MOLT-4 and MOLT-17 cell lines. CONCLUSION: In conclusion, dihydroartemisinin demonstrates anti-tumor activities in human ALL cells via inhibition of cell survival and growth. Dihydroartemisinin augments the apoptotic effect of ABT-737 by inhibiting the expression of Mcl-1.

The Effects of ABT-199 and Dihydroartemisinin Combination on Cell Growth and Apoptosis in Human U937 and KG-1 Cancer Cells.

INTRODUCTION: Change in the balance of Bcl-2 family proteins is one of the main reasons for resistance of tumor cells to ABT-199. In this study, the effect of dihydroartemisinin on cell growth, apoptosis and sensitivity of the AML cells to ABT-199 was investigated. METHODS: Cell proliferation and survival were assessed by trypan blue staining and MTT assay, respectively. Cell apoptosis was measured by Hoechst 33342 staining and caspase-3 activity assay. The expression levels of Bcl-2, Mcl-1 and Bax mRNA were tested by qRT-PCR. RESULTS: Our data showed that combination therapy significantly reduced the IC50 value and synergistically decreased the AML cell survival and growth compared with dihydroartemisinin or ABT-199 alone. Treatment with each of ABT-199 or dihydroartemisinin alone clearly enhanced the Bax mRNA expression and inhibited the expression of Mcl-1 and Bcl-2 mRNA. Inhibition of Mcl-1 mRNA by dihydroartemisinin was associated with enhancement of apoptosis induced by ABT-199 in AML cells. CONCLUSION: In conclusion, dihydroartemisinin not only triggers the intrinsic pathway of apoptosis, but also can increase the sensitivity of the AML cells to ABT-199 via suppression of Mcl-1 expression.

Resveratrol ameliorates spermatogenesis by increasing protamine 1, 2 and HSPA2 expression in experimental varicocele rat model / El resveratrol mejora la espermatogénesis al aumentar la expresión de protamina 1, 2 y HSPA2 en un modelo experimental de ratas con varicocele

Objectives: Varicocele is a common cause of male infertility associated with an elevated testicular temperature that induces apoptosis, spermatogenesis dysfunction, and affects sperm parameters. In this study, we investigate the probable therapeutic effects of resveratrol (RES), a natural phytoalexin, against varicocele. Materials and methods: In this study, 48 male Wistar rats randomly divided into 8 groups: normal, sham, normal+RES (20 and 50mg/kg), varicocele, varicocele+ethanol and varicocele+RES (20 and 50mg/kg). Incomplete closure of the left renal vein was used for varicocele induction and two months later, RES was administrated orally for 60 days. Then, sperm parameters, DNA fragmentations, chromatin density, and testis histopathology were analyzed. In addition, HSPA2, protamine 1, and 2 expression levels were evaluated using real-time PCR. Results: According to our results, resveratrol treatment improved sperm parameters, testis histopathology, DNA fragmentation, and chromatin maturation which damaged follow varicocele (p≤0.05). Also, it increased HSPA2, protamine 1, and 2 expression levels significantly in both doses (p≤0.05). Conclusion: Resveratrol potentially attenuates varicocele-induced spermatogenic impairments by its antioxidant features and regulates spermatogenic gene expression undergoing DNA fragmentation, so leads histopathological properties of tissues to physiological parameters. (AU)

Objetivos: El varicocele es una causa común de infertilidad masculina asociada con una temperatura testicular elevada que induce apoptosis, disfunción de la espermatogénesis y afecta los parámetros espermáticos. En este estudio, investigamos los probables efectos terapéuticos del resveratrol (RES), una fitoalexina natural, contra el varicocele. Materiales y métodos: En este estudio, 48 ratas Wistar macho se dividieron aleatoriamente en 8 grupos: normal, simulado, normal+ RES (20 y 50mg/kg), varicocele, varicocele+ etanol y varicocele+ RES (20 y 50mg/kg). Se utilizó el cierre incompleto de la vena renal izquierda para la inducción del varicocele y dos meses después se administró RES por vía oral durante 60 días. Luego se analizaron los parámetros espermáticos, las fragmentaciones de ADN, la densidad de cromatina y la histopatología testicular. Además, los niveles de expresión de HSPA2, protamina 1 y 2 se evaluaron mediante PCR en tiempo real. Resultados: Según nuestros resultados, el tratamiento con resveratrol mejoró los parámetros espermáticos, la histopatología testicular, la fragmentación del ADN y la maduración de la cromatina que dañó el varicocele posterior (p≤0,05). Además, aumentó significativamente los niveles de expresión de HSPA2, protamina 1 y 2 en ambas dosis (p≤0,05). Conclusión: El resveratrol atenúa potencialmente las deficiencias espermatogénicas inducidas por varicocele por sus características antioxidantes y regula la expresión de genes espermatogénicos que sufren fragmentación del ADN, por lo que conduce las propiedades histopatológicas de los tejidos a parámetros fisiológicos. (AU)

Triggering of Endoplasmic Reticulum Stress by Tannic Acid Inhibits the Proliferation and Migration of Colorectal Cancer Cells.

INTRODUCTION: Due to the pivotal role of endoplasmic reticulum (ER) stress in cancers, interfering with its function can cause the accumulation of unfolded proteins, which ultimately leads to the activation of the unfolded protein response (UPR) signaling pathway and apoptosis. Therefore, the use of plant compounds such as tannic acid with UPR-inducing properties can be proposed as a possible treatment method for cancer. In this study, we investigated the effect of tannic acid on cell migration, colony formation, growth, and UPR-induced apoptosis in the SW48 colorectal cancer cell line. METHODS: The MTT assay was performed to investigate the cytotoxic effect of tannic acid. We performed the qPCR method to elucidate the effect of tannic acid on the expression of Bim, MMP-9, Bcl-xL, cyclin D1, CHOP, and ATF4 genes. We also used the colony formation and migration experiments to investigate the effect of this compound on the colony formation and migration ability of tumor cells. Finally, we used Hoechst staining to measure cell apoptosis. RESULTS: Tannic acid inhibited the cell survival, clonogenic, and migration of colon cancer cells. This compound increased the expression of ER stress-mediated UPR genes, ATF4 and CHOP. Moreover; tannic acid increased the expression of pro-apoptotic proteins like Bim, while at the same time causing a sharp decline in the expression of anti-apoptotic protein Bcl-xL. A decline in MMP-9 expression confirmed the anti-metastatic role of this compound. CONCLUSION: Taken together, tannic acid can induce apoptosis via ER stress-mediated UPR pathway, and has a suppressive effect on cell viability, growth, migration, colony formation, and metastasis, suggesting it may be a potential drug in colorectal cancer treatment.

Resveratrol ameliorates spermatogenesis by increasing protamine 1, 2 and HSPA2 expression in experimental varicocele rat model.

OBJECTIVES: Varicocele is a common cause of male infertility associated with an elevated testicular temperature that induces apoptosis, spermatogenesis dysfunction, and affects sperm parameters. In this study, we investigate the probable therapeutic effects of resveratrol (RES), a natural phytoalexin, against varicocele. MATERIALS AND METHODS: In this study, 48 male Wistar rats randomly divided into 8 groups: normal, sham, normal+RES (20 and 50mg/kg), varicocele, varicocele+ethanol and varicocele+RES (20 and 50mg/kg). Incomplete closure of the left renal vein was used for varicocele induction and two months later, RES was administrated orally for 60 days. Then, sperm parameters, DNA fragmentations, chromatin density, and testis histopathology were analyzed. In addition, HSPA2, protamine 1, and 2 expression levels were evaluated using real-time PCR. RESULTS: According to our results, resveratrol treatment improved sperm parameters, testis histopathology, DNA fragmentation, and chromatin maturation which damaged follow varicocele (p≤0.05). Also, it increased HSPA2, protamine 1, and 2 expression levels significantly in both doses (p≤0.05). CONCLUSION: Resveratrol potentially attenuates varicocele-induced spermatogenic impairments by its antioxidant features and regulates spermatogenic gene expression undergoing DNA fragmentation, so leads histopathological properties of tissues to physiological parameters.

Induction of Unfolded Protein Response by Tannic Acid Triggers Apoptosis in MDA-MB-231 Breast Cancer Cells.

INTRODUCTION: Endoplasmic reticulum (ER) stress can reduce cell survival and enhances the apoptosis of cancer cells. Plant polyphenols like tannic acid trigger ER stress and apoptosis and therefore can be a novel agent for the treatment of cancer. In this study, we investigated the effect of tannic acid on survival, migration, colony formation, ER stress pathway, and apoptosis of the MDA-MB-231 breast cancer cells. METHODS: The MTT assay was performed to investigate the effect of tannic acid on the cell survival of breast cancer cells. We used the qPCR method to reveal the effect of tannic acid on the Bak, CHOP, ATF4, P21, MMP-2, and Bcl-2 expression. Also, colony formation, cell migration, and Hoechst staining assays were employed. RESULTS: The results of the MTT test showed that tannic acid reduced the cell survival rate. In the qPCR assay, we found that tannic acid decreased the expression levels of MMP-2, Bcl-2, ATF4, and CHOP genes, paradoxically, enhanced the expression of Bak and P21 genes. The colony formation and cell migration assays indicated that tannic acid significantly diminished breast cancer cell proliferation and migration, respectively. In the apoptosis assay, tannic acid increased the number of apoptotic cells. CONCLUSION: Tannic acid increases the rate of cell death but decreases viability and cell migration. Moreover, tannic acid induces apoptosis in breast cancer cells. Overall, our study demonstrates that tannic acid induces ER stress by increasing the genes which are playing role in ER stress pathway. These results show that tannic acid can be used as an effective agent for breast cancer treatment.

Evaluating the Magnolol Anticancer Potential in MKN-45 Gastric Cancer Cells.

Background and Objectives: Combination therapy improves the effect of chemotherapy on tumor cells. Magnolol, used in treating gastrointestinal disorders, has been shown to have anti-cancer properties. We investigated the synergistic effect of cisplatin and magnolol on the viability and maintenance of MKN-45 gastric cancer cells. Materials and Methods: The toxicity of magnolol and/or cisplatin was determined using the MTT technique. The trypan blue method was used to test magnolol and/or cisplatin's effect on MKN-45 cell growth. Crystal violet staining was used to assess the treated cells' tendency for colony formation. The expression of genes linked to apoptosis, cell cycle arrest, and cell migration was examined using the qPCR method. Results: According to MTT data, using magnolol and/or cisplatin significantly reduced cell viability. The ability of the treated cells to proliferate and form colonies was also reduced considerably. Magnolol and/or cisplatin treatment resulted in a considerable elevation in Bax expression. However, the level of Bcl2 expression was dramatically reduced. p21 and p53 expression levels were significantly increased in the treated cells, while MMP-9 expression was significantly reduced. Conclusions: These findings show that magnolol has a remarkable anti-tumor effect on MKN-45 cells. In combination with cisplatin, magnolol may be utilized to overcome cisplatin resistance in gastric cancer cells.

MiRNA-16-1 Suppresses Mcl-1 and Bcl-2 and Sensitizes Chronic Lymphocytic Leukemia Cells to BH3 Mimetic ABT-199.

Objective: Chronic lymphoid leukemia (CLL) is the most common type of leukemia among adults. Increased levels of Mcl-1 and Bcl-xL is linked to resistance to Bcl-2 inhibitors including ABT-199. In this study, we investigated the effect of miRNA-16-1 on apoptosis and sensitivity of the CLL cells to ABT-199.
Materials and Methods: In this experimental study, the Mcl-1 and Bcl-2 expression were measured using qualitative reverse transcription-polymerase chain reaction (qRT-PCR) and western blotting. The effect of treatments on cell survival and growth were explored with MTT assay and Trypan blue assay, respectively. The drug interaction was evaluated using combination index analysis. Apoptosis was assessed by ELISA cell death and caspase-3 activity assays.
Results: MiRNA-16-1 markedly inhibited the expression of Mcl-1 and Bcl-2 in a time dependent manner (P<0.05, relative
to blank control). Pretreatment with miRNA-16-1 synergistically suppressed the cell growth and survival and reduced the half-maximal inhibitory concentration (IC50) value of ABT-199. Moreover, miRNA-16-1 markedly augmented the apoptotic effect of ABT-199 in CLL cells (P<0.05).
Conclusion: Our findings propose that miRNA-16-1 act in concert with ABT-199 to exert synergistic anticancer efficacy against CLL, which is attributed to the inhibition of Bcl-2 and Mcl-1. This may propose a promising strategy for CLL resistant patients.

Dual Targeting of Anti-Apoptotic Proteins Enhances Chemosensitivity of the Acute Myeloid Leukemia Cells.

BACKGROUND: Acute myeloid leukemia (AML) is a type of blood cancer characterized by fast cellular proliferation. Myeloid cell leukemia-1 (Mcl-1) and survivin, as anti-apoptotic proteins, are involved in cancer growth and resistance to chemotherapy. The aim of this study was to examine the combination effect of Mcl-1 and survivin specific siRNAs on chemosensitivity of the human HL-60 AML cells. METHODS: SiRNAs transfection was performed by using Lipofectamine™2000 reagent. The mRNA expression was analyzed by real-time quantitative PCR. The apoptosis analysis was measured by ELISA cell death assay. RESULTS: siRNAs markedly suppressed mRNA expression levels of Mcl-1 and survivin in a time-dependent manner, resulting in reduction of leukemic cell proliferation and enhanced spontaneous cell death. Surprisingly, Mcl-1 siRNA and survivin siRNA synergistically enhanced the cell toxic effects of etoposide. Furthermore, down-regulation of Mcl-1 and survivin significantly enhanced the apoptotic effect of etoposide. CONCLUSIONS: Our investigation suggests that suppression of Mcl-1 and survivin by siRNA can effectually inhibit cell growth and overcome chemoresistance of AML cells. Therefore siRNAs may be an important adjuvant in chemotherapy for AML patients.

Combination of two miRNAs has a stronger effect on stimulating apoptosis, inhibiting cell growth, and increasing erlotinib sensitivity relative to single miRNA in A549 lung cancer cells.

Despite the dramatic efficacy of EGFR-TKIs, most of non-small cell lung cancer patients ultimately develop resistance to these agents. In this study, we explored the effects of miRNA-125a-5p and miRNA-145, alone or in combination, EGFR expression, cell growth and sensitivity of the NSCLC cells to erlotinib. The expression of EGFR was measured using RT-qPCR and Western blotting. The effect of miRNAs and erlotinib on cell growth and survival was assessed by trypan blue assay and MTT assay, respectively. Apoptosis was measured using ELISA cell death assay. We found that transfection of miRNA-125a-5p and miRNA-145 significantly inhibited the expression of EGFR mRNA and protein in a time-dependent manner (p < 0.05 vs. blank control or negative control miRNA). ANOVA and Bonferroni's test were used to ascertain significant differences between groups. Other experiments indicated that upregulation of each of miRNA-125a-5p or miRNA-145 inhibited cell growth, induced apoptosis, and markedly decreased the IC50 value of erlotinib in A549 lung cancer cells (p < 0.05). Moreover, the combination of two miRNAs showed a stronger effect on cells survival, apoptosis, and drug sensitivity, relative to single miRNA (p < 0.05). The results of our study indicate that the therapeutic delivery of miRNA-145 and miRNA-125a-5p to lung cancer may inhibit cell proliferation, trigger apoptosis, and sensitize lung cancer cells to EGFR-TKIs.

Combination Therapy with PIK3R3-siRNA and EGFR-TKI Erlotinib Synergistically Suppresses Glioblastoma Cell Growth In Vitro.

BACKGROUND: Up-regulation of PIK3R3 (Phosphoinositide-3-Kinase Regulatory Subunit 3), the regulatory subunit of PI3K is correlated with the drug resistance of the glioblastoma cells. In the present study, the effect of PIK3R3 siRNA on erlotinib sensitivity of the U373-MG glioblastoma cells was explored. METHODS: After PIK3R3 siRNA transfection, the expression of PIK3R3 mRNA was measured using RT-qPCR. Trypan blue exclusion assay was used to explore the effect of PIK3R3 siRNA on cell proliferation. The effects of PIK3R3 siRNA and erlotinib, alone and in combination, on cell survival and apoptosis were measured using MTT assay and ELISA cell death assay, respectively. RESULTS: Our data showed that PIK3R3 siRNA markedly suppressed the expression of PIK3R3 in a time dependent way, inhibited the proliferation of the U373-MG cells and triggered apoptosis (p <0.05, relative to blank control). Pretreatment with PIK3R3 siRNA synergistically decreased the cell survival rate and lowered the IC50 of erlotinib. Moreover, PIK3R3 siRNA markedly enhanced the apoptotic effect of erlotinib. CONCLUSIONS: Our data propose that suppression of PIK3R3 can effectively triggers apoptosis and enhances the sensitivity of the glioblastoma cells to EGFR-TKI erlotinib. Thus, PIK3R3 can be a potential therapeutic target in glioblastoma patients.
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Coenzyme Q10 Modulates Apoptotic Effects of Chronic Administration of Methadone on NMRI Mouse Hippocampus.

OBJECTIVE: Methadone is one of the widely used drug substances prescribed in treatment of opioid dependence and pain management; however, several studies have shown its neurotoxic effects on individuals and animal models. The purpose of this study was to assess neuroprotective effects of Coenzyme Q10 (CoQ10) on neurotoxicity induced by methadone in hippocampus of adult NMRI male mice. MATERIALS AND METHODS: In this experimental study, 48 adult NMRI male mice were randomly divided into 4 groups (n=12 in each) including Methadone, Methadone with sesame oil, Methadone with CoQ10 and saline. The injections of methadone, saline and sesame oil were performed intraperitoneally for 20 days. 24 hours after last injection, half of the animals in each group (n=6) were randomly assessed for evaluating of spatial memory by radial maze. Following behavioral study, animals were sacrificed, and their brains were removed to evaluate pyknotic cells through histological assessment. The remaining were used to study the expression of Arc, Bax, Bcl-2 and Bdnf genes. RESULTS: Results of the present study showed that daily administration of methadone increased the number of pyknotic neurons in the CA1 hippocampus and altered the expression of Bax, Bdnf, Arc and Bcl-2. However, it did not alter spatial memory comparing to saline group. CoQ10 treatment significantly reduced the number of pyknotic cells and expression of Bax, Bdnf, Arc when compared to the vehicle group treated by sesame oil. However, the expression of Bcl-2 significantly increased as a result of CoQ10 treatment. CONCLUSION: CoQ10 reduced the neuronal damage caused by methadone in the hippocampus CA1.

Knockdown of Myeloid Cell Leukemia-1 by MicroRNA-101 Increases Sensitivity of A549 Lung Cancer Cells to Etoposide.

Background: Studies have shown that myeloid cell leukemia-1 (Mcl-1) is the target gene for microRNA -101 (miRNA-101), and decreased levels of miRNA-101 are associated with elevated levels of Mcl-1 and lung cancer survival. The objective of the present study was to investigate the effect of miRNA-101 on the sensitivity of A549 lung cancer cells to etoposide. Methods: The study was conducted during 2018 and 2019 at Arak University of Medical Sciences, Arak, Iran. The effect of miRNA-101 on Mcl-1 expression was assessed using reverse transcription-quantitative polymerase chain reaction 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), and trypan blue exclusion assays were performed to determine the effect of treatments on cell survival and proliferation, respectively. The interaction between miRNA-101 and etoposide was evaluated using the combination index analysis of Chou-Talalay. Apoptosis was quantified using ELISA cell death assay. ANOVA and Bonferroni's tests were used to determine statistical differences between the groups (P<0.05). GraphPad Prism software (version 6.01) was used for data analysis. Results: The results showed that miRNA-101 clearly inhibited the expression of Mcl-1 and reduced the growth of A549 cells, relative to blank control and negative control miRNA (P<0.05). Transfection of miRNA-101 synergistically enhanced the sensitivity of the A549 cells to etoposide. Apoptosis assay data also showed that miRNA-101 triggered apoptosis and augmented the etoposide-mediated apoptosis. Conclusion: Up-regulation of miRNA-101 inhibited cell survival and proliferation, and sensitized A549 cells to etoposide by suppressing Mcl-1 expression. miRNA-101 replacement therapy can be considered as an effective therapeutic strategy in non-small cell lung cancer.

MiRNA-Mediated Knock-Down of Bcl-2 and Mcl-1 Increases Fludarabine-Sensitivity in CLL-CII Cells.

BACKGROUND: Over-expression of anti-apoptotic proteins such as Bcl-2 and Mcl-1 is associated with resistance to chemotherapeutic agents such as fludarabine. Moreover, an inverse relationship between miRNA-15a levels with Bcl-2 and Mcl-1 expression has been observed in CLL patients. In this study, the effect of miRNA-15a on apoptosis and sensitivity of the CLL cells to fludarabine was investigated. METHODS: After treatments, the Mcl-1 and Bcl-2 expression levels were quantified by RT-qPCR. Trypan blue assay was used to explore the effects of miRNA-15a and fludarabine on cell proliferation. The cytotoxicity was measured using MTT assay and combination index analysis. Cell death was determined using cell death detection ELISA assay and caspase-3 activity assay Kits. RESULTS: Results showed that miRNA-15a clearly decreased the mRNA levels of Bcl-2 and Mcl-1 in a time dependent manner, which led to CLL-II cell proliferation inhibition and enhancement of apoptosis (p < 0.05, relative to control). Transfection of the miRNA-15a synergistically reduced the cell survival rate and lowered the IC50 value of fludarabine. Furthermore, miRNA-15a significantly enhanced the apoptotic effect of fludarabine. CONCLUSIONS: Our data propose that suppression of Bcl-2 and Mcl-1 by miRNA-15a can effectively inhibit the cell proliferation and sensitize CLL cells to fludarabine. Therefore, miRNA-15a can be considered as a potential therapeutic target in CLL resistant patients.

Allium jesdianum hydro alcoholic extract ameliorates diabetic nephropathy by suppressing connective tissue growth factor (CTGF) and receptor for advanced glycation endproducts (RAGE) gene expression in diabetic rats with streptozotocin.

OBJECTIVES: Diabetic nephropathy is one of the major complications of diabetes, the use of medicinal plants is increasing due to fewer side effects. This study was designed to examine antidiabetic effects of Allium jesdianum (A. jesdianum) ethanolic extract and evaluate its effects on oxidative stress markers and the expression of connective tissue growth factor (CTGF) and receptor for advanced glycation endproducts (RAGE) genes in the kidney of type 1 diabetic rats. METHODS: In this study, we randomly divided 24 rats into four groups with six rats in each group as follows: Cnt group: normal control receiving normal saline, Dibt group: diabetic control receiving normal saline daily, Dibt + A. jesdianum 250 group: diabetic rats receiving A. jesdianum at a dose of 250 mg/kg bw daily, Dibt + A. jesdianum 500 group: diabetic rats receiving A. jesdianum at a dose of 500 mg/kg bw daily. To induce diabetes, we used 55 mg/kg bw dose of streptozotocin intraperitoneally. The concentration of fasting blood glucose (FBG) and serum urea, creatinine and albumin, SOD, MDA (using spectrophotometric methods) and gene expression of CTGF and RAGE in kidney tissue (using real-time PCR methods) were quantified in the diabetic rats that received A. jesdianum for 42 days, and were compared to control rats. RESULTS: The results showed that in the diabetic group the FBG and serum urea, creatinine and expression of kidney CTGF and RAGE genes and the levels of SOD and MDA significantly increased and serum albumin significantly decreased compared to the Cnt group (p<0.001). Administration of A. jesdianum significantly improved the FBG and serum urea, creatinine and albumin compared to Dibt group (p<0.05). It was shown the A. jesdianum significantly decrease the kidney expression levels of CTGF and RAGE genes and improve oxidative stress (increased SOD and decreased MDA) in the kidney tissues when compared to Dibt group (p<0.001). Also, it was found that the beneficial effects of the A. jesdianum were dose-dependent. CONCLUSIONS: The results of this study showed that administration of A. jesdianum for 42 days has beneficial anti-diabetic and anti-nephropathic effects in diabetic rats and can be used as an adjunct therapy in the treatment of diabetes.

Effect of Artemisia absinthium ethanolic extract on oxidative stress markers and the TLR4, S100A4, Bax and Bcl-2 genes expression in the kidney of STZ-induced diabetic rats.

OBJECTIVES: The present study was conducted to examine antidiabetic effects of Artemisia absinthium ethanolic extract [A. absinthium] and to investigate its effects on oxidative stress markers and the expression of TLR4, S100A4, Bax and Bcl-2 genes in the kidney of STZ-induced diabetic rats. METHODS: Thirty six rats (weight 200-250 g) were randomly divided into diabetes and control groups. Induction of diabetes was performed using STZ (55 mg/kg.bw). Biochemical parameters and oxidative stress markers (SOD and MDA) were measured using spectrophotometry after 60 days of treatment. The expression of TLR4, S100A4, Bax and Bcl-2 were analyzed by real-time PCR. One-way analysis of variance (ANOVA) and Bonferroni post hoc test were used to compare the data. RESULTS: Diabetes significantly impairs the serum fasting blood glucose (FBG), lipid profile, urea, creatinine and albumin. At the end of treatment with A. absinthium extract, these parameters were close to the normal range. The results showed that the A. absinthium extract significantly decreased the kidney expression of TLR4, S100A4, Bax and increased the expression of Bcl-2 and improved oxidative stress markers (SOD and MDA) in the kidney tissues of treated rats. Also, all of these beneficial effects of the A. absinthium were dose-dependent. CONCLUSIONS: The extract of A. absinthium possesses antidiabetic effects. A. absinthium decreased the expression of TLR4, S100A4, Bax and increased the expression of Bcl-2 and improved oxidative stress. Therefore, this herbal extract can be used as an adjuvant treatment for diabetic complications.

MiRNA-7 Replacement Effect on Proliferation and Tarceva-Sensitivity in U373-MG Cell Line.

BACKGROUND: Deregulation of the EGFR signaling pathway activity has been shown to can be effective in resistance to EGFR-TKIs, such as Tarceva (erlotinib), in glioblastoma cells. In addition, reports have shown that the reduction of miRNA-7 expression levels is associated with an increase in the expression of EGFR. Here, we evaluated the effect of miRNA-7 on EGFR expression and sensitivity of the U373-MG glioblastoma to erlotinib. METHODS: The effect of miRNA-7 on EGFR expression was examined using RT-qPCR and western blotting. Trypan blue and MTT assays were performed to explore the effect of treatments on cell growth and survival, respectively. The combination index analysis was used to evaluate the interaction between drugs. Apoptosis was measured by ELISA cell death assay. RESULTS: We showed that miRNA-7 markedly inhibited the expression of EGFR and decreased the growth of glioblastoma cells, relative to blank control and negative control miRNA (p < 0.05). Introduction of miRNA-7 synergistically increased the sensitivity of the U373-MG cells to erlotinib. Results of apoptosis assay demonstrated that miRNA-7 can trigger apoptosis and enhance the erlotinib-mediated apoptosis. CONCLUSIONS: Our results show that miRNA-7 plays a critical role in the growth, survival and sensitivity of the U373-MG cells to erlotinib by targeting EGFR. Thus, miRNA-7 replacement therapy can become an effective therapeutic procedure in glioblastoma.