Oleanolic acid increases the anticancer potency of doxorubicin in pancreatic cancer cells

Combination therapy is a novel cancer therapy approach that combines two or more chemotherapy drugs. This treatment modality enhances the efficacy of chemotherapy by targeting key pathways in an additive or synergistic manner. Therefore, we investigated the efficacy of combination therapy by widely used chemotherapy drug doxorubicin (DOX) and oleanolic acid (OA) to induction of apoptosis for pancreatic cancer (PC) therapy. The effects of DOX, OA, and their combination (DOX-OA) were investigated on proliferation and viability of PC cell line (PANC-1) by MTT assay. Moreover, migration and invasion of the cancer cells were evaluated by trans-well migration assay and wound healing assay. Flow cytometry and DAPI (4′,6-diamidino-2-phenylindole) staining were employed to investigate apoptosis quantification and qualification of the treated cancer cells. Finally, mRNA expression of apoptosis-related genes was assessed by quantitative real-time polymerase chain reaction. Our results demonstrated that the proliferation and metastasis potential of PC cells significantly decreased after treatment by DOX, OA, and DOX-OA. Moreover, we observed an increase in apoptosis percentage in the treated cancer cells. The apoptosis-related gene expression was modified to increase the apoptosis rate in all of the treatment groups. However, the anticancer potency of DOX-OA combination was significantly more than that of DOX and OA treatments alone. Our study suggested that DOX-OA combination exerts more profound anticancer effects against PC cell lines than DOX or OA monotherapy. This approach may increase the efficiency of chemotherapy and reduce unintended side effects by lowering the prescribed dose of DOX.     

The response of extracellular signal-regulated kinase (ERK) to androgen-induced proliferation in the androgen-sensitive prostate cancer cell line,LNCaP

The mechanisms by which androgens stimulate proliferation of prostate cancer cells are poorly understood. It has been proposed that androgen stimulation may induce the mitogen-activated protein (MAP) kinase system in prostate cancer cells and lead to cellular proliferation. We attempted to evaluate the role of the extracellular signal-regulated kinase (ERK) pathway in the stimulation by androgens of prostate cancer cell proliferation. Androgen-sensitive prostate cancer cell line (LNCaP) cells plated on sterile glass coverslips were treated with 10^?8 M dihydrotestosterone (DHT) or epidermal growth factor (EGF) (10 ng/ml) for periods ranging from 1 min to 96 h. The proliferative index of the cells, evaluated by immunoperoxidase staining of cells with an antibody to Ki-67, was increased at least two-fold at all time points from 5 min to 48 h following exposure to either DHT or EGF. Immunohistochemical evaluation of ERK1/2 and pERK (activated ERK) demonstrated high levels of ERK1/2 in untreated LNCaP cells, while pERK was expressed at much lower levels. Following treatment with DHT, no change in staining intensity for either ERK1/2 or pERK was observed, while treatment with EGF resulted in no change in ERK1/2, but significantly increased cytoplasmic staining for pERK at all time points beyond 2 min. These results were confirmed by Western blot analysis of ERK1/2 and pERK expression in these cell lines following treatment with DHT or EGF. Our findings suggest that the proliferative response of prostate cancer cells to androgens, unlike the proliferative response to EGF, is not mediated by the activation of ERK1/2, and that currently undefined pathways other than those involving ERK1/2 are involved.     

Biomarkers of Residual Disease,Disseminated Tumor Cells,and Metastases in the MMTV-PyMT Breast Cancer Model

Cancer metastases arise in part from disseminated tumor cells originating from the primary tumor and from residual disease persisting after therapy. The identification of biomarkers on micro-metastases, disseminated tumors, and residual disease may yield novel tools for early detection and treatment of these disease states prior to their development into metastases and recurrent tumors. Here we describe the molecular profiling of disseminated tumor cells in lungs, lung metastases, and residual tumor cells in the MMTV-PyMT breast cancer model. MMTV-PyMT mice were bred with actin-GFP mice, and focal hyperplastic lesions from pubertal MMTV-PyMT;actin-GFP mice were orthotopically transplanted into FVB/n mice to track single tumor foci. Tumor-bearing mice were treated with TAC chemotherapy (docetaxel, doxorubicin, cyclophosphamide), and residual and relapsed tumor cells were sorted and profiled by mRNA microarray analysis. Data analysis revealed enrichment of the Jak/Stat pathway, Notch pathway, and epigenetic regulators in residual tumors. Stat1 was significantly up-regulated in a DNA-damage-resistant population of residual tumor cells, and a pre-existing Stat1 sub-population was identified in untreated tumors. Tumor cells from adenomas, carcinomas, lung disseminated tumor cells, and lung metastases were also sorted from MMTV-PyMT transplant mice and profiled by mRNA microarray. Whereas disseminated tumors cells appeared similar to carcinoma cells at the mRNA level, lung metastases were genotypically very different from disseminated cells and primary tumors. Lung metastases were enriched for a number of chromatin-modifying genes and stem cell-associated genes. Histone analysis of H3K4 and H3K9 suggested that lung metastases had been reprogrammed during malignant progression. These data identify novel biomarkers of residual tumor cells and disseminated tumor cells and implicate pathways that may mediate metastasis formation and tumor relapse after therapy.     

A comparative study on the activity and antigenicity of truncated and full-length forms of streptokinase

Application of streptokinase (SK) as a common and cost-effective thrombolytic drug is limited by its antigenicity and undesired hemorrhagic effects. Prior structural/functional and epitope-mapping studies on SK suggested that removal of 59 N-terminal residues led to its fibrin dependency and identified SK antigenic regions, respectively. Following in silico analyses two truncated SK proteins were designed and compared for their fibrin specificity and antigenicity with the full-length SK. Computer-based modeling was used to predict the effect of vector (pET41a)-born protein tags on the conformation of SK fragments. SK60-386, SK143-386 and full-length SK (1-414) were separately cloned, expressed in BL21 E. coli cells and confirmed by Western-blotting. Functional activity of the purified proteins was evaluated with chromogenic and clot lysis assays and their antigenicity was tested by ELISA assay using rabbit anti-streptokinase antibody. As expected, chromogenic bioassay showed a major activity decline for SK60-386 and SK143-386 (83 and 91 percent, respectively), compared to SK1-414. However, in clot lysis assay, which is a fibrin-dependent pharmacopoeia-approved test, SK60-386 and SK143-386 were respectively 35 and 31 percent more active though lysed the clots slower than full-length SK. Antigenic analysis also indicated significant decrease in ELISA signals obtained for truncated proteins compared to SK1-414 (45 and 28 percent less reactivity for SK143-386 and SK60-386, respectively, p < 0.0001). The results of this study for the first time pointed to SK143-386 and SK60-386, as improved SK derivatives with increased fibrin-selectivity and decreased antigenicity as well as acceptable bioactivity profiles in a pharmacopoeia-based analysis, which deserve more detailed pharmacological studies.     

Cytokine gene polymorphisms and susceptibility to cutaneous leishmaniasis in Iranian patients

Cutaneous Leishmaniasis (CL) is one of the most prevalent clinical forms of leishmaniasis. Preliminary data suggest that cytokine gene polymorphisms can contribute to resistance or susceptibility to CL. Therefore, we investigated the association of functional polymorphisms in four cytokine genes with susceptibility to, and clinical outcome of CL. A total of 201 patients with self-healing cutaneous leishmaniasis (SCL) and 92 asymptomatic infected controls (AIC) from Fars province as well as 58 patients with chronic cutaneous leishmaniasis (CCL) and their 688 normal controls (normal Iranian population or NIP) who were collected from the different areas of Iran were included in the study. The allele-specific oligonucleotide polymerase chain reaction (ASO-PCR) or PCR-RFLP (restriction fragment length polymorphism) methods were used for genotyping. The frequency of TNF-alpha -308 G-->A and TNF-beta +252 G-->A gene polymorphisms were not different between studied groups. Distribution of IFN-gamma +874 A-->T and IL-4 -590 C-->T polymorphism were also compared between SCl or CCL patients and their controls. IFN-gamma +874 A-->T polymorphism was less common in CCL patients compared to the NIP (chi(2)=12.53, p=0.0019). Significant differences in frequency of IL-4 -590 C -->T polymorphism were also found between the SCL and AIC (chi(2)=8.64, p=0.003). In conclusion, our results suggest that functional genetic variants in the IL-4 promoter could influence the risk of developing CL while the polymorphism in the first intron of the IFN-gamma gene might influence the progression of disease towards CCL.     

Application of Soil‐borne Actinomycetes for Biological Control against Fusarium Wilt of Chickpea (Cicer arietinum) caused by Fusarium solani fsp pisi

Black root rot, caused by Fusarium solani f.sp. pisi, is a devastating soil‐borne disease in chickpea in Iran with no effective control measures. With the aim of finding applicable biocontrol agents to alleviate the malady, isolates of Actinomycetes isolated from soil and their antagonistic effect against F. solani f.sp. pisi were evaluated both in vitro and in vivo. More than 100 Actinomycetes isolates were screened for their antifungal activities against the pathogen. The most active isolates were evaluated in greenhouse for their biocontrol performance. Based on the results of dual cultures in screening evaluations, the size of inhibition zone of fungal growth, and the most effective antagonist isolates (S3, S12 and S40) were selected for further studies. Identity of active isolates was determined, in this regard, 16S rDNA of isolates were amplified using universal bacterial primers FD1 and RP2. The PCR products were purified and sequenced. Sequence analysis of 16S rDNA was then performed using NCBI BLAST method. Comparison of the near full length 16S rRNA sequence of isolates to GenBank sequences demonstrated that isolates S3 and S12 were most similar to Streptomyces antibioticus, while isolate S40 was most similar to Streptomyces peruviensis. Biocontrol studies of these isolates in control of the disease in greenhouse significantly decreased the disease severity. Actinomycetes isolate S12 demonstrated the greatest effect in reducing disease than the other two. Results of this research are at preliminary stage for developing biocontrol agents. These data can be utilized as a platform for future studies with the aim of commercializing these biocontrol products and hoping to step towards sustainable agriculture.     

Utility of DNA barcoding to identify rare endemic vascular plant species in Trinidad

The islands of the Caribbean are considered to be a “biodiversity hotspot.” Collectively, a high level of endemism for several plant groups has been reported for this region. Biodiversity conservation should, in part, be informed by taxonomy, population status, and distribution of flora. One taxonomic impediment to species inventory and management is correct identification as conventional morphology‐based assessment is subject to several caveats. DNA barcoding can be a useful tool to quickly and accurately identify species and has the potential to prompt the discovery of new species. In this study, the ability of DNA barcoding to confirm the identities of 14 endangered endemic vascular plant species in Trinidad was assessed using three DNA barcodes (matK, rbcL, and rpoC1). Herbarium identifications were previously made for all species under study. matK, rbcL, and rpoC1 markers were successful in amplifying target regions for seven of the 14 species. rpoC1 sequences required extensive editing and were unusable. rbcL primers resulted in cleanest reads, however, matK appeared to be superior to rbcL based on a number of parameters assessed including level of DNA polymorphism in the sequences, genetic distance, reference library coverage based on BLASTN statistics, direct sequence comparisons within “best match” and “best close match” criteria, and finally, degree of clustering with moderate to strong bootstrap support (>60%) in neighbor‐joining tree‐based comparisons. The performance of both markers seemed to be species‐specific based on the parameters examined. Overall, the Trinidad sequences were accurately identified to the genus level for all endemic plant species successfully amplified and sequenced using both matK and rbcL markers. DNA barcoding can contribute to taxonomic and biodiversity research and will complement efforts to select taxa for various molecular ecology and population genetics studies.