Monomethyl auristatin E, a potent cytotoxic payload for development of antibody-drug conjugates against breast cancer

Background: Breast cancer is a heterogeneous disease characterized by differential responses to targeted and chemotherapeutic agents. Antibody-drug conjugates are one of the promising strategies for the treatment of breast cancer. Monomethyl auristatin E [MMAE] is a highly potent microtubule inhibitor and a common payload used for development of antibody-drug conjugates. The purpose of this study was to investigate the cytotoxic effects of MMAE on breast cancer cell lines

Materials and Methods: MDA-MB-468 and MDA-MB-453 cells were treated with MMAE at various concentrations [1, 10, 100, and 1000 ng/ml], and cytotoxicity was measured after 48 and 72 hours using an MTT assay

Results: Our findings indicated that MMAE possesses dose- and time-dependent cytotoxic activities against human breast cancer cells. The morphological features of the treated cells were supportive of the cytotoxic activity of MMAE. The results of the MTT assay showed that MMAE has a significant cytotoxicity against MDA-MB-468 and, to a lesser degree, MDA-MB-453 cells

Conclusion: MMAE can be used as a highly cytotoxic payload for development of antibody-drug conjugates against breast cancer

In vitro therapeutic effects of low level laser at mrna level on the release of skin growth factors from fibroblasts in diabetic mice

Numerous in vitro reports suggest that Low Level Laser Therapy [LLLT] affects cellular processes by biostimulation, however most of them emphasize on using visible light lasers which have low penetration. The aim of this study was to determine the effect of infrared laser light [which is more useful in clinic because of its higher penetration] on secretion of Fibroblast Growth Factor [FGF], Platelet Derived Growth Factor [PDGF] and Vascular Endothelial Growth Factor [VEGF], as important growth factors in wound healing. Fibroblasts were extracted from the skin of 7 diabetic and 7 nondiabetic mice and cultured. Cell cultures of experimental group were irradiated with single dose of LLLT [energy density of 1 J/ cm[2]] using an 810 nm continuous wave laser and the control group was not irradiated. Secretion of growth factors by skin fibroblasts were quantified through real time polymerase chain reaction. Diabetic irradiated group showed significant increase in FGF [p=0.017] expression, although PDGF increased and VEGF decreased in both diabetic and nondiabetic irradiated groups, but these variations were not statistically significant. These results suggest that LLLT may play an important role in wound healing by stimulating the fibroblasts

Association of HLA-DRB1, DQA1 and DQB1 alleles and haplotypes with common variable immunodeficiency in iranian patients

Common Variable Immunodeficiency [CVID] is an antibody deficiency syndrome that often co-occurs in families with selective IgA deficiency [IgAD]. This study was designed to investigate the frequency of DR and DQ loci of HLA class II region in common variable immunodeficiency [CVID] patients. Fifteen Iranian patients with CVID or IgAD [mean age 14.6 +/- 5.4, range 4-25 years; 9 male and 6 female] and 63 healthy controls were studied. Establishment of B-lymphoblastoid cell lines was performed using Epstein-Barr-virus [EBV] immortalization technique and HLA alleles were typed using polymerase chain reaction based on sequence specific primers [PCR-SSP]. DRB1 alleles including DRB1 *04 [p=0.03] and DRB1 *11 [p=0.01] significantly showed higher frequency in the studied subjects. In contrast, DRB1 *301 [p=0.04] and DRB1 *07 [p=0.02] alleles were negatively associated with CVID. For DQB1 and DQA1 loci, DQB1 *0302 [p=0.047] and DQA1 *03011 [p=0.001] demon-strated high frequency in cases, while DQB1 *0201 [p=0.02] and DQA1 *0201 [p=0.01] were detected to be low when compared to controls. Haplotype analysis indicated that frequency of DRB1*04-DQB1*03011-DQA1 *03011 [p=0.02], DRB1 *11-DQB1 *03011-DQA1 *0505 [p=0.047], DRB1 *11-DQA1 *0505 [p=0.04] and DRB1*04-DQA1*03011 [p=0.02] haplotypes were significantly higher in patient group, while only the frequency of the DRB1 *07-DQA1 *0201 haplotype gene was statistically lower in control group [p=0.02]. According to the results, it could be deduced that the HLA-DR and DQ loci may contribute to the pathogenesis of CVID or they might be considered as suitable markers for the possibility of the occurrence of this genetic defect

Efficacy of HPV-16 E7 based vaccine in a TC-1 tumoric animal model of cervical cancer

The human papillomavirus as an etiological agent of cervical cancer does not grow adequately in tissue culture systems. The tumor cell line TC-1 continuously expresses the E6 and E7 oncogenic proteins of HPV, and is considered a suitable tool in laboratory investigations and vaccine researches against cervical cancer The TC-1 cell line was grown in RPMI 1650 supplemented with 10% FBS, glutamine and antibiotics, and was used for tumor development in mice. Six to seven week-old tumor bearing C57BL/6 mice were divided into 3 groups consisting of 7 mice per group. The first group received pcDNA-E7, the second group received pcDNAS, and the third group received phosphate buffered saline [PBS]. The treated animals were monitored for their tumor size progression and survival. At last, the tumoric tissues from autopsied animals were fixed and examined with Mayer's hematoxylin and eosin [H and E]. All experiments were done in accordance with guidelines of the Laboratory Animal Ethical Commission of Tarbiat Modares University. Data analysis was performed using the oneway ANOVA followed by Tukey's test in both experimental and control groups. A p-value <0.05 was considered significant. There were significant decreases in tumor growth; there were also improvements in survival among mice in the treated groups [p<0.041]. H and E stained sections from untreated mice were studied independently in a blinded fashion by two observers and showed malignant neoplasms composed of severely pleomorphic tumor cells with nuclear enlargement, high nuclear-cytoplasmic [N/C] ratios, and prominent nucleoli in solid and fascicular patterns of growth. High mitotic activity with extensive necrosis was also noted in both test and control groups. The TC-1 lung metastatic model can be used to test the efficacy of various E7-based therapeutic cancer vaccine strategies for cervical cancer and the prevention of HPV-related neoplasia

[Up regulation of CD82 gene in prostate cancer PC-3 cell line treated with silibinin]

Prostate cancer is one of the most common cancer in the developed countries. Most of cancer deaths are due to development of metastasis. Hence, prevention of metastasis is critical. Silibinin is a flavonoid component that inhibits cell proliferation and causes cell death of human prostate cancer. In this study, the expression of CD82 gene in PC-3 cells treated with escalating concentrations of silibinin was evaluated which can result in new view for prostate cancer therapy. In this study, PC-3 cells were treated with different concentrations of silibinin for 24h. The LD50 was determined. RNA was extracted by trizol, then cDNA was synthesized. Precise primers were designed for CD82 and GAPDH genes by specific software. Quantity of CD82 gene expression compare to GAPDH gene in different concentrations of silibilin was analyzed using very sensitive quantitative Real-time PCR. CD82 gene expression in PC-3 cells treated with 100, 150 and 200?g/ml of silibinin at 24h was increased by 1.97 +/- 0.26 [P<0.05], 3.00 +/- 0.26 and 3.43 +/- 0.43 [P<0.01], respectively. The results of quantitative Real-time PCR indicated that silibinin can probably decrease metastasis, by up-regulation of CD82 metastasis suppressor gene in PC-3 cells

[HIV type 1-based particles effect in transduction of HT1080 cell line: a technique in beta-thalassemia gene therapy]

One of the most effective methods in the treatment of beta-thalassemia is gene therapy by viral vectors. The aim of this study was to design a recombinant lentivirus containing mini LCR and beta-globin gene for transferring normal beta-globin gene into hematopoetic stem cells. In this basic-applied study, each segment was cloned into a lenti transfer vector and confirmed by restriction digestion and sequencing. Transfer vector and three packaging plasmids were cotransfected into 293T packaging cells using lipofectamine 2000. Harvested viruses were confirmed by RT PCR on extracted RNA of these recombinant lentiviruses. The titer of lentiviral stock was determined in a HT1080 cell line. Transduction of target cells was increased by polybrene until 2 fold. Transduced HT1080 colonies remained after 2-week antibiotic selection. The remained transduced HT1080 colonies were expanded and DNA was extracted. PRC evaluated random integration of construct into the genome in this gene transfer technique. PCR evaluated random integration of construct into the genome in this gene transfer technique. Optimum MOI for HT1080 cell line was determined. Lenti viruses can be used for effective and permanent gene transferring in mammalian cells such as hematopoetic stem cells in order to accomplish gene therapy of genetic diseases like beta thalassemia and cancers

HLA-DRB1, DQA1 and DQB1 alleles and haplotypes frequencies in Iranian healthy adult responders and non-responders to recombinant hepatitis B vaccine

Different studies have demonstrated that a small proportion of healthy individuals receiving the hepatitis B [HB] vaccine do not produce protective levels of anti-HB antibody, a phenomenon which could be linked to certain human leukocyte antigen [HLA] class-II alleles or haplotypes. The present study was undertaken to determine the frequency of HLA class-II alleles in Iranian healthy adult responders and non-responders to HB vaccine. Twelve non-responders [anti-HBs antibody < 10 IU/L] and 46 responders [anti-HBs antibody > 100 IU/L] were tissue typed for HLA class-II. HLA-DRB1, DQB1 and DQA1 alleles were determined using polymerase chain reaction based on sequence specific primers [PCR-SSP] technique. Accessibility to excess amount of genomic DNA was possible using Epstein-Barr virus [EBV]-transformed B-cells established from all vaccinees. Our results demonstrated increased frequencies of HLA- DRB1*07, DRB1*03, DRB1*04, DQB1*0201, DQA1*0201 alleles and HLA- DRB1*07/DQB1*0201/DQA1*0201 and DRB1*04/DQB1*0302/DQA1*03011 haplotypes in the non-responder group. Comparison between responders and non-responders revealed only a significant difference for DQB1*0201 allele [p < 0.05]. These findings confirm the association of certain HLA alleles and haplotypes with the lack of antibody response to HB vaccine in an Iranian population

Transduction of COS-7 and K562 cell lines by recombinant lentiviral particles carrying miniLCR and B-globin gene: Introduction to gene therapy of major B-thalassemia

Beta-thalassemia is caused by absence of reduction of beta-globin chain synthesis. One of the effective therapeutic methods for this disease can be gene therapy by viral vectors. The capacity of lentiviral vectors is approximately 8 kb, we designed a 6 kb construct containing mini LCR and beta-globin gene instead of LCR region. The aim of this study is to make a recombinant lentiviruses containing miniLCR and beta-globin gene for transfer to the target cells for gene therapy of beta-thalassemia. HS2, HS3, HS4 segments [miniLCR] and beta-globin gene with 5' and 3' UTR were amplified from the genomic DNA of a normal individual by PCR. Each segment was cloned in pTZ57R/T vector and then sub cloned first into the pBGGT vector and finally into the pLenti-Dest vector. Final transfer vector and the three helper packaging plasmids [Plp1, Plp2 Plp/VSVG] were contransfected into 293T packaging cells using lipofectamine 2000. Harvested viruses were confirmed by RT-PCR on extracted RNA of these recombinant lentiviruses. The titer of lentiviral stock determined in a K562 cell line and compared with COS-7 cell line. The titer in both cell lines was the same. Optimum MOI for COS-7 cell line was 5 and when polybrene was used transduction increased by 2 fold. The remaining transduced COS-7 colonies were expanded and DNA was extracted. By PCR, random intergration construct into the genome was evaluated. The produced lentiviruses can be an appropriate means for effective transfer of the designed construct into dividing and non-dividing cells such as hematopoetic stem cells for transplantation of beta thalassemia patients. Efficiency of transduction by leniviruses is more than the gene targeting technique. Also units of HS2, HS3 and HS4 regions in mini LCR and selection of larger HS3 unit may increase the expression of beta globin gene