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.

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.

Hydroalcoholic extract of Scrophularia striata has a significant therapeutic effect on thioacetamide-induced liver cirrhosis in rats.

OBJECTIVES: Liver cirrhosis is one of the most important causes of death from liver diseases. Nowadays, the use of herbal medicines has increased due to its availability, less side effects and cheapness for the treatment of liver diseases. The present study was conducted to examine therapeutic effects of hydroalcoholic extract of Scrophularia striata (S. striata) on thioacetamide-induced liver cirrhosis in rats through evaluate its effects on oxidative stress markers and the expression of metalloproteinase 1 (TIMP 1), toll-like receptor-4 (TLR-4), and Mitofusin (MFN2) genes. METHODS: 24 male rats were selected by simple random sampling. Rats were randomly assigned to four groups: group I: healthy rats, group II: thioacetamide (TAA) injected rats, group III: TAA injected rats+100 mg/kg bw of S. striata and group IV: TAA injected rats+200 mg/kg bw of S. striata. Liver cirrhosis was induced in rats by a 300 mg/kg bw TAA administration twice with an interval of 24 h. After 8 weeks of treatment by S. striata at doses of 100 and 200 mg/kg bw, biochemical factors and oxidative stress markers (SOD, TAC, GPX, CAT and MDA) were measured using spectrophotometric methods. Also, gene expression of TIMP 1, TLR-4, and MFN2 were analyzed using real-time PCR. ANOVA and Bonferroni post hoc test analysis were applied to evaluate the data. RESULTS: The results showed the S. striata extract significantly improve the serum ALT, AST and ALP levels, TIMP 1, TLR-4, and MFN2 genes and oxidative stress markers (SOD, TAC, GPX, CAT and MDA) in the liver tissues when compared to control group (p<0.05). Also, it was found that the beneficial effects of the S. striata were dose-dependent. CONCLUSIONS: Based on the results obtained S. striata by reducing the expression of TIMP 1, TLR-4, and MFN2 genes and improving oxidative stress might be used as adjuvant treatment for liver cirrhosis.

The investigation of the frequency of the alpha-1-antitrypsin phenotype in patients with liver cirrhosis.

OBJECTIVES: Alpha-1-antitrypsin (AAT) has different phenotypes. Evidence suggests that the abundance of each of these phenotypes may be associated with a disease. The purpose of this study was to evaluate the frequency of AAT phenotypes in patients with liver cirrhosis as well as in healthy individuals. METHODS: In this study, 42 patients with liver cirrhosis were selected. The results of the previous research done by the researcher on healthy individuals were used to construct the control group. After obtaining informed consent, 5 mL of fasting venous blood sample was taken, and phenotypes were analyzed by isoelectric focusing. Data were analyzed using Chi-square and Fisher's exact tests at a significant level of 0.05. RESULTS: The results of this study indicated that all 42 healthy subjects had an MM allele (100%). However, among 42 patients, 35 (83.3%) had an MM allele, 5 (11.9%) had an MS allele, and 2 (4.8%) had MZ allele. The difference between the two groups was significant (p=0.02). There was no difference between men and women in the allele type (p=0.557). CONCLUSIONS: This study revealed that MS and MZ alleles were observed only in patients with liver cirrhosis, and none of these alleles were found in healthy subjects. Therefore, MS and MZ alleles can be further investigated as risk factors for liver cirrhosis.

Anticonvulsive and anti-epileptogenesis effects of Echinacea purpurea root extract, an involvement of CB2 receptor.

OBJECTIVES: Phytocannabinoids beyond the Δ9-tetrahy-drocannabinol have shown anticonvulsive effects. Also, alkylamides from Echinacea purpurea have been proved as cannabinomimetics. We examined the effect of the hydroalcoholic root extract of E. purpurea on pentylenetetrazol (PTZ)-induced tonic-clonic seizures and kindling model of epileptogenesis and the involvement of CB2 receptors as the mediator of this effect. METHODS: Male Wistar rats (200 ± 20 g) were used. Single intraperitoneal (i.p.) injection of PTZ (80 mg/kg) was used to induce tonic-clonic seizures. The kindling model of epileptogenesis was induced by daily injections of PTZ (37 mg/kg; i.p. for 15 days). Latency and duration of the stages were monitored for analysis. The hydroalcoholic root extract of E. purpurea was injected (i.p.) 20 min before seizure induction at the doses of 10, 50, 100 and 200 mg/kg. CB2 receptor antagonist SR144528 was injected (0.1 mg/kg; i.p.) 20 min before the Echinacea injection. RESULTS: In the tonic-clonic model, pretreatment with E. purpurea at the doses of 100 and 200 mg/kg significantly increased latencies to S2-S6, while it significantly decreased S6 duration and mortality rate. SR144528 injection before the injection of 100 mg/kg of E. purpurea significantly prevented the effects of the extract on S4-S6 latencies. In the kindling model, E. purpurea at the doses of 100 and 200 mg/kg significantly delayed epileptogenesis and decreased mortality rate, while SR144528 injection before the injection of 100 mg/kg of E. purpurea significantly blocked this effect of the extract. CONCLUSIONS: These findings revealed the anticonvulsive and antiepileptogenesis effects of the E. purpurea root extract, which can be mediated by CB2 receptors.

Biochanin-A has antidiabetic, antihyperlipidemic, antioxidant, and protective effects on diabetic nephropathy via suppression of TGF-ß1 and PAR-2 genes expression in kidney tissues of STZ-induced diabetic rats.

One of the major complications of diabetes is diabetic nephropathy, and often many patients suffer from diabetic nephropathy. That is why it is important to find the mechanisms that cause nephropathy and its treatment. This study was designed to examine the antidiabetic effects of biochanin A (BCA) and evaluate its effects on oxidative stress markers and the expression of transforming growth factor-ß1 (TGF-ß1) and protease-activated receptors-2 (PAR-2) genes in the kidney of type 1 diabetic rats. After induction of diabetes using streptozotocin (STZ), 55 mg/kg bw dose, rats were randomly divided into four groups with six rats in each group as follows: normal group: normal control receiving normal saline and a single dose of citrate buffer daily; diabetic control group: diabetic control receiving 0.5% dimethyl sulfoxide daily; diabetic+BCA (10 mg/kg) group: diabetic rats receiving biochanin A at a dose of 10 mg/kg bw daily; diabetic+BCA (15 mg/kg) group: diabetic rats receiving biochanin A at a dose of 15 mg/kg bw daily. TGF-ß1 and PAR-2 gene expression was assessed by real-time. Spectrophotometric methods were used to measure biochemical factors: fast blood glucose (FBG), urea, creatinine, albumin, lipids profiles malondialdehyde (MDA), and superoxide dismutase (SOD). The course of treatment in this study was 42 days. The results showed that in the diabetic control group, FBG, serum urea, creatinine, expression of TGF-ß1 and PAR-2 genes, and the levels of MDA in kidney tissue significantly increased and SOD activity in kidney tissue and serum albumin significantly decreased compared to the normal group (p < 0.001). The results showed that administration of biochanin A (10 and 15 mg/kg) after 42 days significantly reduced the expression of TGF-ß1 and PAR-2 genes and FBG, urea, creatinine in serum compared to the diabetic control group (p < 0.001), also significantly increased serum albumin compared to the diabetic control group (p < 0.001). The level of MDA and SOD activity in the tissues of diabetic rats that used biochanin A (10 and 15 mg/kg) was significantly reduced and increased, respectively, compared to the diabetic control group (p < 0.001). Also, the result showed that in the diabetic control group lipids profiles significantly is disturbed compared to the normal group (p < 0.001), the results also showed that biochanin A (10 and 15 mg/kg) administration could significantly improved the lipids profile compared to the control diabetic group (p < 0.001). It is noteworthy that it was found that the beneficial effects of the biochanin A were dose dependent. In conclusion, administration of biochanin A for 42 days has beneficial effect and improves diabetes and nephropathy in diabetic rats. So probably biochanin A can be used as an adjunct therapy in the treatment of diabetes.

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.

Gene therapy: A promising approach for breast cancer treatment.

Breast cancer (BC) is the most prevalent malignancy and the second leading cause of death among women worldwide that is caused by numerous genetic and environmental factors. Hence, effective treatment for this type of cancer requires new therapeutic approaches. The traditional methods for treating this cancer have side effects, therefore so much research have been performed in last decade to find new methods to alleviate these problems. The study of the molecular basis of breast cancer has led to the introduction of gene therapy as an effective therapeutic approach for this cancer. Gene therapy involves sending genetic material through a vector into target cells, which is followed by a correction, addition, or suppression of the gene. In this technique, it is necessary to target tumour cells without affecting normal cells. In addition, clinical trial studies have shown that this approach is less toxic than traditional therapies. This study will review various aspects of breast cancer, gene therapy strategies, limitations, challenges and recent studies in this area.

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.