Evaluation of Epidermal Growth Factor Receptor Gene Mutations in an Iranian Population with Non-Small Cell Lung Carcinoma.

Background: Mutations of the epidermal growth factor receptor (EGFR) gene, predominantly in exons 18-21, have been highlighted to function as the crucial predictors of the response rate of patients with non-small cell lung cancer (NSCLC) to EGFR tyrosine kinase inhibitors (TKIs). Methods: This study was performed at Tehran University of Medical Sciences. Data and information were retrospectively collected from the period between Dec 2010 and Apr 2014. Exons 18 to 21 of the EGFR were analyzed for any potential mutation by PCR, accompanied by DNA sequencing on 160 with pathological confirmation of NSCLC. Results: Demographically, the male to female ratio was approximately 2:1, and a substantial difference in age between sexes was not observed (P=0.065), but a noticeable difference was found in the smoking variable, where 77.8% of males were smokers compared to 17.3% of women (odds ratio (OR) (95% CI) = 16.72 (7.15-39.11)). We found a frequency of 10.63% (17/160) for mutations found in exons 19 and 21, nonetheless, no mutations in exon 18 and exon 20 were observed. The most frequently observed mutations were c.2235_2249, del and c.2240_2257, del in exon 19 and p. L858R in exon 21. The c.2253A>G was found as a novel mutation that was the rarest mutation detected in this work. Interestingly, a remarkable negative association was revealed between smoking and mutation rates in NSCLC patients (OR (95% CI) = 0.13 (0.04-0.46). Conclusion: The occurrence of EGFR mutations is largely varied among the different states of Iran, probably due to variations in ethnicity, smoking rate, and sex ratio of participants.

Overexpression of Iron Super Oxide Dismutases A/B Genes Are Associated with Antimony Resistance of Leishmania tropica Clinical Isolates.

Background: Pentavalent antimonial has been a drug of choice against leishmaniasis, despite the emergence of treatment failure. Identification of resistance markers is urgently needed to design new therapeutic strategies. Iron-Superoxide dismutases (Fe-SODs) are antioxidant enzymes contributing to detoxify reactive oxygen species to prevent a cell from oxidative stress. Since antimonial compounds induce oxidative stress, in this survey, the expression of SOD genes was investigated to identify their expression pattern in clinical resistant isolates. Methods: This cross-sectional survey was done in Mashhad City, northeast of Iran during 2014 to 2019. The RNA expression level of mitochondrial (SODA) and glycosomal (SODB) superoxide dismutase was investigated in 25 antimony responsive (n=15) and unresponsive (n=10) anthroponotic cutaneous leishmaniasis (ACL) patients. Total RNA extraction and cDNA synthesis, the qRT-PCR approach was utilized to investigate the relative RNA expression level. Results: The transcript level of SODs was over-expressed in the most resistant isolates. Gene expression analysis demonstrated the over-expression of SODA and B by a factor of 3.8 and 4.81, respectively, in resistance isolates vs. sensitive ones. Conclusion: Aberrant expression of SODA/B in unresponsive parasites could potentially implicate in detoxifying antimony-induced oxidative stress. Moreover, SODs might be considered as potential predictive markers of the response to antimonials in ACL patients in endemic areas.

Identification of immunodominant proteins of Leishmania infantum by immunoproteomics to evaluate a recombinant multi-epitope designed antigen for serodiagnosis of human visceral leishmaniasis.

Visceral leishmaniasis (VL) is a protozoan disease caused by Leishmania infantum in the Mediterranean region including Iran. In 95% of cases, the disease can be fatal if not rapidly diagnosed and left untreated. We aimed to identify immunoreactive proteins of L. infantum (Iranian strain), and to design and evaluate a recombinant multi-epitope antigen for serodiagnosis of human VL. To detect the immunoreactive proteins of L. infantum promastigotes, 2DE immunoblotting technique was performed using different pooled sera of VL patients. The candidate immunoreactive proteins were identified using MALDI-TOF/TOF mass spectrophotometry. Among 125 immunoreactive spots detected in 2-DE gels, glucose-regulated protein 78 (GRP78), ubiquitin-conjugating enzyme E2, calreticulin, mitochondrial heat shock 70-related protein 1 (mtHSP70), heat shock protein 70-related protein, i/6 autoantigen-like protein, ATPase beta subunit, and proteasome alpha subunit 5 were identified. The potent epitopes from candidate immunodominant proteins including GRP78, mtHSP70 and ubiquitin-conjugating enzyme E2 were then selected to design a recombinant antigenic protein (GRP-UBI-HSP). The recombinant antigen was evaluated by ELISA and compared to direct agglutination test for detection of anti L. infantum human antibodies. We screened 34 sera of VL patients from endemic areas and 107 sera of individuals without L. infantum infection from non-endemic area of VL. The recombinant protein-based ELISA provided a sensitivity of 70.6% and a specificity of 84.1%. These results showed that GRP78, ubiquitin-conjugating enzyme E2, and mtHSP70 proteins are potential immunodominant targets of the host immune system in response to the parasite and they can be considered as potential candidate markers for diagnosis purposes.

Gene expression analysis of antimony resistance in Leishmania tropica using quantitative real-time PCR focused on genes involved in trypanothione metabolism and drug transport.

Pentavalent antimonials remain the treatment of choice for all the clinical forms of leishmaniasis. The increasing rates of antimony resistance are becoming a serious health problem in treatment of anthroponotic cutaneous leishmaniasis (ACL). Accordingly, unraveling molecular markers is crucial for improving medication strategies and monitoring of drug-resistant parasites. Different studies have suggested the importance of genes involved in trypanothione metabolism and drug transport. In this regard, present study was designed to investigate the RNA expression level of five genes including γ-GCS, ODC, TRYR (involved in trypanothione metabolism), AQP1 (acts in drug uptake) and MRPA (involved in sequestration of drug) in sensitive and resistant Leishmania tropica isolates. Seven antimony-resistant and seven antimony-sensitive L. tropica clinical isolates were collected from ACL patients. Drug sensitivity test was performed on the samples as well as reference strains; afterwards, gene expression analysis was performed on clinical isolates by quantitative real-time PCR. The results revealed that the average expression level of AQP1 gene was decreased (0.47-fold) in resistant isolates compared to sensitive ones whereas MRPA (2.45), γ-GCS (2.1) and TRYR (1.97) was upregulated in resistant isolates. The average expression of ODC (1.24-fold) gene was not different significantly between sensitive and resistant isolates. Our findings suggest that AQP1, MRPA, GSH1 and TRYR can be considered as potential molecular markers for screening of antimony resistance in some L. tropica clinical isolates.

Effect of photodynamic therapy based on indocyanine green on expression of apoptosis-related genes in human gingival fibroblast cells.

BACKGROUND: Periodontal diseases refer to inflammation of the gingiva, induction of apoptosis in human gingival fibroblast cells, destruction of the surrounding tissues, and early bone loss resulting in infections due to the pathogenic activity of the microorganisms and the host immune inflammatory responses. Recent investigations have suggested that antimicrobial photodynamic therapy (aPDT) can be an adjunct treatment therapy for periodontal infections. AIM: To prove the lack of side effects of PDT on periodontal tissues, we investigated the expression of BAX and BCL-2 genes that are involved in apoptosis after the PDT on human gingival fibroblast (HGF) cells. MATERIALS AND METHODS: In this study the effect of PDT based on indocyanine green (ICG) as a photosensitizer with the diode laser were tested on the expression of BAX and BCL-2 genes in monolayers of HGF cells. The effects of PDT on the expression of BAX and BCL-2 genes were evaluated by real-time quantitative reverse transcription PCR. RESULTS: The results of the genes expression analysis revealed that ICG-PDT at concentrations 1000µg/mL, induced the significant expression of BAX in HGF cells; however, the laser irradiation as well as ICG showed no significant effects on the expression of these genes. Treatment with ICG alone, laser irradiation and ICG-PDT caused no observable BCL-2 gene expression changes between the tested and control groups. CONCLUSION: Our findings indicate that ICG-PDT at 1000µg/mL of ICG with the exposure time of 60s for the diode laser would appear to be an inducer of apoptosis in HGF at transcriptome level.

Effects of sub-lethal doses of photo-activated disinfection against Porphyromonas gingivalis for pharmaceutical treatment of periodontal-endodontic lesions.

BACKGROUND: Microorganisms treated by photo-activated disinfection (PAD) in combined periodontal-endodontic (perio-endo) lesions would be exposed to sub-lethal doses of PAD (sPAD). This study evaluated the effect of sPAD using toluidine blue O (TBO) in combination with diode laser irradiation on the growth and biofilm-formation ability of Porphyromonas gingivalis as an endo-periodontal pathogen. METHODS: The antibacterial and antibiofilm potential of sPAD against P. gingivalis was analyzed at sub-lethal doses of TBO and irradiation time of diode laser on a colony-forming unit and crystal violet assays, respectively. RESULTS: TBO-mediated PAD, using 6.25-100µg/mL at a fluency of 171.87J/cm2 and 12.5-100µg/mL at a fluency of 137.5J/cm2, showed a significant dose-dependent reduction in P. gingivalis growth when compared to the control. TBO-mediated PAD showed a significantly inhibitory effect on biofilm formation in P. gingivalis than TBO-PAD at sub-lethal levels. CONCLUSION: High doses of sPAD revealed antibacterial and antibiofilm potential activity, whereas lower doses of sPAD had conflicting results. Therefore, when PAD is prescribed in combined perio-endo lesions treatment, the dose of PAD used in vivo should be taken into account.

Detection and molecular identification of leishmania RNA virus (LRV) in Iranian Leishmania species.

Leishmania RNA virus (LRV) was first detected in members of the subgenus Leishmania (Viannia), and later, the virulence and metastasis of the New World species were attributed to this virus. The data on the presence of LRV in Old World species are confined to Leishmania major and a few Leishmania aethiopica isolates. The aim of this study was to survey the presence of LRV in various Iranian Leishmania species originating from patients and animal reservoir hosts. Genomic nucleic acids were extracted from 50 cultured isolates belonging to the species Leishmania major, Leishmania tropica, and Leishmania infantum. A partial sequence of the viral RNA-dependent RNA polymerase (RdRp) gene was amplified, sequenced and compared with appropriate sequences from the GenBank database. We detected the virus in two parasite specimens: an isolate of L. infantum derived from a visceral leishmaniasis (VL) patient who was unresponsive to meglumine antimoniate treatment, and an L. major isolate originating from a great gerbil, Rhombomys opimus. The Iranian LRV sequences showed the highest similarities to an Old World L. major LRV2 and were genetically distant from LRV1 isolates detected in New World Leishmania parasites. We could not attribute treatment failure in VL patient to the presence of LRV due to the limited number of specimens analyzed. Further studies with inclusion of more clinical samples are required to elucidate the potential role of LRVs in pathogenesis or treatment failure of Old World leishmaniasis.

Evaluation of photo-activated disinfection effectiveness with methylene blue against Porphyromonas gingivalis involved in endodontic infection: An in vitro study.

BACKGROUND: Eradication or suppression of microbial pathogens is a major goal in endodontic infection therapy. Sub-lethal doses of photo-activated disinfection (sPAD) as a new treatment method might be able to control the microorganisms involved in endodontic infections normally treated with PAD. This study evaluated the effect of sPAD using methylene blue (MB) in combination with diode laser irradiation on the growth and biofilm formation ability of Porphyromonas gingivalis as an endodontic pathogen. MATERIALS AND METHODS: The anti-microbial and anti-biofilm potential of sPAD against P. gingivalis were assessed at sub-lethal doses of MB and irradiation by diode laser on colony forming unit and crystal violet assays, respectively. RESULTS: MB-sPAD using 25µg/mL at a fluency of 117.18J/cm2 and 50-100µg/mL at a fluency of 93.75J/cm2 significantly P. gingivalis growth when compared to the control. MB at 100µg/mL at a fluency of 117.18J/cm2 in MB-mediated PAD showed a significant inhibitory effect on biofilm formation in P. gingivalis compared with MB-sPAD. CONCLUSION: High doses of MB-mediated sPAD exhibited anti-microbial and anti-biofilm potential activity, whereas lower doses of MB-mediated sPAD did not display this ability. Therefore, the dose of PAD used in vivo should be taken into account for endodontic treatment.

Effects of probiotic Lactobacillus acidophilus and Lactobacillus casei on colorectal tumor cells activity (CaCo-2).

BACKGROUND: The probiotic microorganisms are live normal flora that provide nutritional benefits. When probiotic administered in adequate amounts, they also confer a health benefit on the host. Different mechanisms of probiotic effects include the following: stimulating the immune system, modifying the composition of normal intestinal flora and preventing the carcinogenic activity of fecal enzymes. In this study, direct effects of probiotic lactobacilli on tumor cells were investigated. METHODS: Supernatants and bacterial extracts of two standard Lactobacillus species (L. acidophilus and L. casei) were prepared and CaCo-2 cells were treated with them. Probiotic effects on cell proliferation, necrosis, apoptosis, migration and invasion were assessed. RESULTS: The supernatants of Lactobacilli decreased cell proliferation and increased cell apoptosis, however, no significant effect on cell necrosis was reported. In contrast, Lactobacilli extract, reduced cell proliferation and increased cell apoptosis. Lactobacilli extract also led to cell necrosis. Furthermore, both supernatants and cell extracts of the probiotic agents resulted in decreased cells' migration and invasion. CONCLUSION: In this study, it was shown that Lactobacilli probiotics useful effects are not confined to the enhancement of the immune system; however, they effectively suppress the malignant phenotypes of colorectal cancer cells.

BMI1 and TWIST1 downregulated mRNA expression in basal cell carcinoma.

BACKGROUND: BMI1, TWIST1 and SNAI2/SLUG have been implicated in aggressive behavior of squamous cell carcinoma (SCC) and melanoma and BMI1 expression could identify subtypes of Merkel cell carcinoma (MCC). However, BMI1, TWIST1 and SNAI2 expression levels in basal cell carcinomas (BCCs) have not been elucidated. We hypothesized BCC could be a good model system to decipher mechanisms which inhibit processes that drive tumor metastasis. The aim of this study was to examine the mRNA expression level of BMI1, TWIST1, and SNAI2 in BCCs. MATERIALS AND METHODS: Thirty-five fresh non-metastatic BCC tissue samples and seven fresh normal skin tissue samples were evaluated by real-time RT-PCR. RESULTS: BMI1 and TWIST1 demonstrated marked down-regulation (p<0.00l, p=0.00l respectively), but SNAI2 showed no significant change (p=0.12). CONCLUSIONS: Previous literature has clearly demonstrated a positive association between BMI1 and TWIST1 expression and metastatic BCC, aggressive SCC and melanoma. Here, we demonstrated a negative association between BMI1 and TWIST1 mRNA expression level and BCC.

The multidrug resistance pumps are inhibited by silibinin and apoptosis induced in K562 and KCL22 leukemia cell lines.

Silibinin have been introduced for several years as a potent antioxidant in the field of nutraceuticals. Based on wide persuasive effects of this drug, we have decided to investigate the effects of silibinin on chronic myelogenous leukemia (CML) in vitro models, K562 and KCL22 cell lines. Lactate dehydrogenase (LDH) release, microculture tetrazolium test (MTT assay) and real-time PCR were employed to evaluate the effects of silibinin on cell cytotoxicity, cell proliferation and expression of various multidrug resistance genes in these cell lines, respectively. Our results have shown that presence of silibinin has inhibitory effects on cell proliferation of K562 and KCL22 cell lines. Also, our data indicated that silibinin, in a dose-dependent manner with applying no cytotoxic effects, inhibited cell proliferation and reduced mRNA expression levels of some transporter genes e.g. MDR1, MRP3, MRP2, MRP1, MRP5, MRP4, ABCG2, ABCB11, MRP6 and MRP7. The multifarious in vitro inhibitory effects of silibinin are in agreement with growing body of evidence that silibinin would be an efficient anticancer agent in order to be used in multi-target therapy to prevail the therapeutic hold backs against CML.

Differences in vaginal lactobacilli composition of Iranian healthy and bacterial vaginosis infected women: a comparative analysis of their cytotoxic effects with commercial vaginal probiotics.

BACKGROUND: Vaginal flora of healthy women is dominated by Lactobacillus species which can prevent bacterial vaginosis. OBJECTIVES: The current study aimed to determine the differences in vaginal lactobacilli composition of Iranian healthy and bacterial vaginosis (BV) infected women and compared their cytotoxic effects with commercial vaginal probiotics. PATIENTS AND METHODS: One hundred and seventy eight vaginal specimens were collected from healthy and BV infected women. Lactobacillus colonies were obtained by culturing on laked blood BHI and MRS medias and genetically defined by 16s rRNA sequencing. Differentiating the specimens to normal, intermediate and BV infected were carried out by Ison and Hey grading protocol. Identification of Lactobacillus strains in vaginal specimens were performed by Multiplex PCR. The inhibitory effects of lactobacilli on Hela (tumoral cervical cells) and HNCF-pi52 (normal cervical cells) were conducted by MTT and trypan blue assays. RESULTS: L. crispatus, L. gasseri, L. iners, L. jensenii, L. acidophilus and L. rhamnosus were the most frequently occurring species in vagina of healthy Iranian women. L. crispatus and L. jensenni were significantly higher in the normal than in the BV infected groups. Also the cytotoxic effect of L. crispatus on tumoral cervical cells was higher than other lactobacilli including commercial probiotics. CONCLUSIONS: As L. crispatus and L. jensenni were significantly higher in BV infected women and the cytotoxic effect of L. crispatus on tumoral cervical cells was high, introduction of new probiotics seems necessary.

Identification of novel hypoxia response genes in human glioma cell line a172.

OBJECTIVE(S): Hypoxia is a serious challenge for treatment of solid tumors. This condition has been manifested to exert significant therapeutic effects on glioblastoma multiform or (WHO) astrocytoma grade IV. Hypoxia contributes numerous changes in cellular mechanisms such as angiogenesis, metastasis and apoptosis evasion. Furthermore, in molecular level, hypoxia can cause induction of DNA breaks in tumor cells. Identification of mechanisms responsible for these effects can lead to designing more efficient therapeutic strategies against tumor progression which results in improvement of patient prognosis. Materials and Methods : In order to identify more hypoxia regulated genes which may have a role in glioblastoma progression, cDNA-AFLP was optimized as a Differential display method which is able to identify and isolate transcripts with no prior sequence knowledge. RESULTS: Using this method, the current study identified 120 Transcription Derived Fragments (TDFs) which were completely differentially regulated in response to hypoxia. By sequence homology searching, the current study could detect 22 completely differentially regulated known genes and two unknown sequence matching with two chromosome contig and four sequence matches with some Expressed Sequence Tags (ESTs). CONCLUSION: Further characterizing of these genes may help to achieve better understanding of hypoxia mediated phenotype change in tumor cells.

Differential expression of human homeodomain TGIFLX in brain tumor cell lines.

Glioblastoma is the most common and the most lethal primary brain cancer. This malignancy is highly locally invasive, rarely metastatic and resistant to current therapies. Little is known about the distinct molecular biology of glioblastoma multiforme (GBM) in terms of initiation and progression. So far, several molecular mechanisms have been suggested to implicate in GBM development. Homeodomain (HD) transcription factors play central roles in the expression of genomic information in all known eukaryotes. The TGIFX homeobox gene was originally discovered in human adult testes. Our previous study showed implications of TGIFLX in prostate cancer and azoospermia, although the molecular mechanism by which TGIFLX acts is unknown. Moreover, studies reported that HD proteins are involved in normal and abnormal brain developments. We examined the expression pattern of TGIFLX in different human brain tumor cell lines including U87MG, A172, Daoy and 1321N1. Interestingly, real time RT-PCR and western blot analysis revealed a high level of TGIFLX expression in A172 cells but not in the other cell lines. We subsequently cloned the entire coding sequence of TGIFLX gene into the pEGFP-N1 vector, eukaryotic expression vector encoding eGFP, and transfected into the U-87 MG cell line. The TGIFLX-GFP expression was confirmed by real time RT-PCR and UV-microscopic analysis. Upon transfection into U87 cells, fusion protein TGIFLX-GFP was found to locate mainly in the nucleus. This is the first report to determine the nuclear localization of TGIFLX and evaluation of its expression level between different brain tumor cell lines. Our data also suggest that TGIFLX gene dysregulation could be involved in the pathogenesis of some human brain tumors.

Induction of Apoptosis and Growth Suppression by Homeobox Gene TGIFLX in Prostate Cancer Cell Line Lncap.

BACKGROUND: TGIFLX, a Homoproteins cluster member located on the X chromosome, has a critical role in male reproduction and prostate development. Previous studies have shown the erratic expression of TGIFLX gene in a large proportion of prostate tumors. However TGIFLX function in prostate development remains unknown. The purpose of this study was to evaluate the consequences of TGIFLX expression on prostate cancer cell lines (LNCaP). METHOD: Inducible Tet-On gene expression system was used with a regulatory capability by doxycycline induction. In this system, stable LNCaP cells with TGIFLX tet-on plasmid were able to induce TGIFLX expression by doxycycline treatment. TGIFLX gene expression was confirmed by RT-PCR. RESULTS: Induction of gene expression caused cell proliferation decrement and apoptosis increment in LNCaP TGIFLX cells compared with control cells (P<0.01). Also, by using PEGFPN1 plasmid protein in this study localization was shown in nucleus. The gene was cloned in the plasmid and transfected to LNcap cells with plasmid PEGFPN1 TGIFLX and the plasmid was PEGFPN1. The TGIFLX expression was confirmed by RT-PCR and fluorescent microscopy. CONCLUSION: TGIFLX expression demonstrated a tumor suppressor characterization in a prostatic cancer cell line with low grade of tumorigenicity (LNCaP). More cell lines with different level of tumorogenicity need to be investigated for further clarification of the TGIFLX gene function.

Mutation screening in the mitochondrial D-loop region of tumoral and non-tumoral breast cancer in Iranian patients.

The mitochondrial DNA (mtDNA) mutations in mitochondrial coding and non coding regions seem to be important in carcinogenesis. The aim of this investigation was to evaluate coding region (mt-tRNA(Phe) and tRNA(Pro)) and non-coding sequence, mitochondrial displacement loop (mtDNA D-loop), in the cancerous and non-cancerous lesions of Iranian patients with breast cancer (BC). Genomic DNA was extracted from 50 breast tumors and surrounding normal tissue pairs as well as from 50 unrelated normal breast tissues from Iranian Kurdish population. Subsequently, PCR amplification was performed using specific primers, and then PCR products were subjected to direct sequencing. 41 genetic variants were identified in mtDNA D-loop among tumoral and non-tumoral tissues but not in tRNA(Phe) and tRNA(Pro) sequences. Our findings indicated that C182T, 194insT, 285insA and 16342delT were just found in BC tumors whereas 302insC, C309T and C16069T found in both tumors and surrounding normal tissues. Although our findings showed that the observed genetic variations were not restricted to breast cancer tissues, some genetic changes were found only in BC tumors. Our results, in agreement with the evidence from earlier studies, confirm that the mtDNA genetic alterations might be implicated in tumor initiation, progression and development.