Suppression of E6 Oncogene Induces Apoptosis in CaSki Cervical Cancer Cells.

OBJECTIVE: The most important casuse of cervical cancer incidence and high mortality rate is infection to the human papillomavirus (HPV). The aim of the present study was to investigate the effect of silencing HPV E6 oncogene on cervical cancer cells using specific siRNAs. MATERIALS AND METHODS: CaSki cervical cancer cells, carrying E6 gene, were cultured and then transfected with E6 targeting siRNAs. The cell viability through suppression of E6 expression was explored using MTT assay. Besides, apoptosis induction was investigated by means of flow cytometry using Annexin / PI staining. The changes in the expression of target genes were examined via  Real-Time PCR. RESULTS: E6 gene silencing caused a significant decrease in the survival rate of CaSki cells through remarkable enhancement of apoptosis induction. Moreover, E6 suppression led to significant upregulation of P53, Bax, Caspase-3, and Caspase-9 mRNA expression while downregulated Bcl-2 expression. Interestingly, it was found that suppression of E6 expression could lead to upregulation of  E5 and E7 expression as a compensatory mechanism for E6 deactivation. CONCLUSION: According to the results of this study, suppression of E6 expression using specific siRNAs could be considered as a therapeutic approach for cervical cancer.

Investigation of the Anti-Cancer Effects of Free and PLGA-PAA Encapsulated Hydroxytyrosol on the MCF-7 Breast Cancer Cell Line.

OBJECTIVE: Breast cancer is the most frequent cancer among women and the most important cause of death. Surgery and chemotherapy are the common treatment of breast cancer, but increasing drug resistance has created many challenges in its treatment. The present study aimed to investigate the anti-cancer function of free and nano-encapsulated hydroxytyrosol on the MCF-7 breast cancer cell line. METHODS: The poly lactide-co-glycolide-co-polyacrylic acid (PLGA-co-PAA) nanoencapsulated Hydroxytyrosol was synthesized, and the MTT assay was performed to evaluate the anti-proliferative and anti-tumor effects of both free and nano-encapsulated Hydroxytyrosol. After the extraction of RNA from the treated and control cancer cells, cDNA synthesis was performed and the expression of P21, P27, and Cyclin D1 genes was evaluated by Real-Time PCR. RESULTS: The results of the study showed that free (12 ppm and 72 hours) and nanoencapsulate (10 ppm and 24 hours) hydroxytyrosol resulted in 50% death (IC50) of the cancer cells and increased by increasing the concentration and time. Also, free and nano-encapsulated hydroxytyrosol increased the expression of P21 and P27 genes and reduced the expression of Cyclin D1 in breast cancer cells. In general, the nanoencapsulated hydroxytyrosol showed more anticancer function than the free hydroxytyrosol. CONCLUSION: The present study illustrated that hydroxytyrosol could lead to cell death in MCF-7 breast cancer by regulating the cell cycle. Also, the nano-encapsulation of Hydroxytyrosol enhanced the Hydroxytyrosol anticancer function by PLGA-co-PAA. However, for more accurate results, further studies on animal models are necessary.

A Comparison of the Anti-Cancer Effects of Free and PLGA-PAA Encapsulated Hydroxytyrosol on the HT-29 Colorectal Cancer Cell Line.

BACKGROUND: Hydroxytyrosol is one of the phenolic compounds of olive oil and can induce anticancer effects on colorectal cancer cells. OBJECTIVE: The aim of the present study was to evaluate the free hydroxytyrosol and nano-capsulated hydroxytyrosol effects on the cell cycle arrest in HT-29 colorectal cancer cell line. METHODS: The nano-capsulated hydroxytyrosol was synthesized in poly lactide-co-glycolide-co-polyacrylic acid (PLGA-PAA) copolymer. MTT assay was performed to evaluate the anti-proliferative and anti-tumor effects of the free hydroxytyrosol and nano-capsulated hydroxytyrosol. Finally, the relative expression of CDKN1A, CDKN1B, and CCND1 genes was evaluated in control and treated colorectal cancer cells by using Real-Time PCR. RESULTS: The obtained results from the MTT assay showed that the cytotoxic effects of the nano-capsulated hydroxytyrosol on the colorectal cancer cell line (IC50= 6PPM) were significantly more than free hydroxytyrosol (IC50= 12PPM) after 72h. Also, nano-capsulated hydroxytyrosol showed more significant effects on the upregulation of CDKN1A and CDKN1B genes and down-regulation of the CCND1 gene in colorectal cancer cells. CONCLUSION: In conclusion, the present study showed that hydroxytyrosol led to the death of colorectal cancer cells through cell cycle arrest. Also, the PLGA-PAA copolymer dramatically caused to increase the cytotoxic effects of the hydroxytyrosol on the colorectal cancer cells.

Detection of t(8;14) c-myc/IgH gene rearrangement by long-distance polymerase chain reaction in patients with diffuse large B-cell lymphoma.

OBJECTIVE/BACKGROUND: Specific chromosomal translocations are found in human leukemias and lymphomas. These translocations are closely related to particular histological and immunological phenotypes. In Burkitt's lymphoma, translocation t(8;14)(q24;q32), which involves the c-myc gene (8q24) and the immunoglobulin heavy-chain (IgH) locus (14q32), accounts for 90-95% of all chromosomal translocations. This translocation can be found in 2-5% of diffuse large B-cell lymphoma (DLBCL). Long-distance polymerase chain reaction (LD-PCR) assays, which can identify oncogene/Ig gene rearrangement, can detect these fusion genes. The objective of this study was to detect t(8;14) c-myc/IgH gene rearrangement by LD-PCR in patients with DLBCL. METHODS: In this study, 54 DLBCL cases were tested by LD-PCR with specific primers. LD-PCR was used for two breakpoints in both the IgH gene (joining region and γ switch region) and the myc gene (Exons 2 and 3). RESULTS: As much as 1.85% of the samples were positive for the γ constant region and Exon 2 of the myc gene. CONCLUSION: LD-PCR can be used for the detection of t(8;14) c-myc/IgH gene rearrangement in patients with DLBCL.

Investigation of the GJB6 Deletion Mutations Del (GJB6-D13s1830) and Del (GJB6-D13s1854) in Iranian Patients with Autosomal-Recessive Non-Syndromic Hearing Loss (ARNSHL)

Hearing loss (HL) is the most common inherited sensory disorder affecting about 1 in 1000 births. The first locus for nonsyndromic autosomal recessive HL is on chromosome 13q11-22. The two genes, GJB2 and GJB6, are closely located on chromosome and are known to be co-expressed in the embryonic cochlea. Deletion mutations involving GJB6 were associated with autosomal-recessive nonsyndromic hearing loss (ARNSHL) and in combination with a GJB2 mutation with digenic ARNSHL. The objective of this study was to screen for the del (GJB6-D13S1830) and del (GJB6-D13s1854) mutations in GJB6 gene in patients with ARNSHL from Iran, using multiplex PCR and direct sequencing methods. Agarose gel electrophoresis and DNA sequencing of amplified fragment of the PCR reaction showed none of the patients was found to carry deletion in GJB6 gene which indicates that these deletions are restricted to certain populations and indicating a founder effect regarding these deletions.

A novel compound heterozygous mutation (35delG, 363delC) in the Connexin 26 gene causes non-syndromic autosomal recessive hearing loss.

Mutations in the Connexin 26 (Cx26) gene are a common cause of hereditary hearing loss in different populations. In the present study, an Iranian patient with bilateral hearing loss underwent molecular analysis for the causative mutation. DNA studies were performed for the Cx26 gene by PCR and sequencing methods. We describe a novel compound heterozygous mutation (35delG, 363delC) in the Cx26 gene that is strongly associated with congenital non-syndromic hearing loss (NSHL).

A Novel Missense Mutation, E1623G, in the Human Factor VIII Gene Associated With Moderate Haemophilia A.

INTRODUCTION: Haemophilia A is the most common inherited X-linked recessive bleeding disorder. The severity of the resultant bleeding diathesis depends on the FVIII levels associated with the mutation. Analysis of carrier state can be made indirectly by DNA linkage analysis or directly by identifying the mutation that leads to the disease. The aim of this study was to identification of the causal mutation of the FVIII gene in a haemophilic patient. CASE REPORT: Our case is a 16-year-old male haemophilia A patient with some symptoms such as recurrent hemarthrosis in left knee. In this study, we used single-stranded conformational polymorphism (SSCP) and conformational sensitive gel electrophoresis (CSGE) methods and direct sequencing to identify the mutation responsible for haemophilia A in our patient. CONCLUSIONS: We reported a novel missense mutation (GAA→GGA), E1623G, in exon 14 of FVIII gene that is associated with moderate haemophilia A. This new mutation was recorded in GenBank (NCBI) with accession number JF916726.1. This study showed that the use of PCR-CSGE and PCR-SSCP may be useful in detecting most of genetic defects such as point mutations of FVIII gene in haemophilic patients.

A Novel De Novo Dominant Mutation in GJB2 Gene Associated with a Sporadic Case of Nonsyndromic Sensorineural Hearing Loss.

Mutations in the GJB2 gene are the most common known cause of hereditary congenital hearing loss. Rapid genomic DNA extraction (RGDE) method was used for genomic DNA extraction. After amplification of coding region of CX26 gene with specific primers, expected PCR products with 724bp length were subjected to direct sequencing in both directions. We describe here a novel heterozygous -T to -C transition at codon 202 (TGC→CGC) of the GJB2 gene in a patient, 40-year-old Iranian woman, which replaces a cysteine with an arginine residue (C202R). The dominant mutation C202R associated with non-syndromic sensorineural hearing loss. This mutation has not previously been described in affected or control samples from other populations investigated for GJB2 mutations, indicating that it is a rare substitution. This dominant mutation was recorded in NCBI GenBank with accession number KF 638275.

A novel mutation (4040-4045 nt. delA) in exon 14 of the factor VIII gene causing severe hemophilia A.

Hemophilia A is an X-linked congenital bleeding disorder caused by Factor VIII deficiency. Different mutations including point mutations, deletions, insertions and inversions have been reported in the FVIII gene, which cause hemophilia A. In the current study, with the use of conformational sensitive gel electrophoresis (CSGE) analysis, we report a novel 1-nt deletion in the A6 sequence at codons 1328-1330 (4040-4045 nt delA) occurring in exon 14 of the FVIII gene in a seven-year-old Iranian boy with severe hemophilia A. This mutation that causes frameshift and premature stop-codon at 1331 has not previously been reported in the F8 Hemophilia A Mutation, Structure, Test and Resource Site (HAMSTeRS) database.

Identification of a novel missense mutation in exon 4 of the human factor VIII gene associated with sever hemophilia A patient.

Hemophilia A is an X-linked congenital bleeding disorder caused by factor VIII deficiency. The factor VIII gene is on the long arm of the X chromosome at Xq28 spans 186 kb and consists of 26 exons. In this study to identify defects in the factor VIII gene, Single-Stranded Conformation Polymorphism (SSCP) analysis was used. A novel missense mutation due to T --> C transition at codon 153 (TGC) of the factor VIII gene which replace a cysteine with an arginine residue, was found in a patient of North-Western of Iran with sever hemophilia A. Direct sequencing of the amplified fragment was performed to confirm the mutation. This study shows that we can use of Polymerase Chain Reaction (PCR) and silver staining of SSCP methods for detecting most of the point mutations causative hemophilia A.