Epithelial to mesenchymal transition is a process in which a cell experiences a loss of epithelial cell characteristics and acquires a more mesenchymal cell phenotype. In cancer, epithelial to mesenchymal transition has been proposed to play an important role during specific stages of tumor progression. The role epithelial to mesenchymal transition and mesenchymal to epithelial transition might play in toxicant-induced urothelial cancer is unknown.+3- and Cd+2-transformed cell lines, the spheroids isolated from these cell lines as well as the tumor heterotransplants that were produced by the injection of the transformed cells into immune compromised mice.Real-time PCR, Western blotting, immuno-histochemistry and immuno-fluorescence were used to determine the expression of E- and N-cadherin in the UROtsa parent, the As+3- and 7 Cd+2- transformed cell lines generated from human urothelial cells (UROtsa). The expression varied within each cell line, with 10% to 95% of the cells expressing N-cadherin. Tumors produced from these cell lines showed no expression of the N-cadherin protein. Spheroids which are made up of putative cancer initiating cells produced from these cell lines showed only background expression of N-cadherin mRNA, increased expression of aldehyde dehydrogenase 1 mRNA and produced tumors which did not express N-cadherin. There was no change in the expression of E-cadherin in the tumors, and the tumors formed by all the As+3 and Cd+2-transformed cell lines and cancer initiating cells stained intensely and uniformly for E-cadherin.This study showed that N-cadherin expression was increased in 6 AsThe finding that the cells expressing N-cadherin gave rise to tumors with no expression of N-cadherin is in agreement with the classical view of epithelial to mesenchymal transition. Epithelial to mesenchymal transition and N-cadherin are associated with dissemination and not with the ability to establish new tumor growth. Mesenchymal to epithelial transition and E-cadherin are viewed as necessary for a cell to establish a new metastatic site. The lack of N-cadherin expression in tumor transplants is consistent with E-cadherin expressing cells “seeding” a site for tumor growth. The study shows that a minority population of cultured cells can be the initiators of tumor growth. The epithelial to mesenchymal transition (EMT) is defined as a process in which a cell experiences a loss of epithelial cell characteristics and acquires a more mesenchymal cell phenotype. In cancer, EMT has been proposed to play an important role during specific stages of tumor progression, such as invasion and intravasation, where tumor cells disassemble and migrate to tissue and/or organ sites distant from the primary tumor –3; howev+3) and cadmium (Cd+2) exposure and the development of urothelial cancer. Exposure to environmental arsenic is strongly associated with urothelial cancer, and high incidences of this cancer are also linked to localities that exhibit increased rates of arsenic-induced skin cancer [+2 exposure due to the high accumulation of this metal seen in smokers [+3 and Cd+2 exposure and the development of urothelial cancer. The cell line, immortalized via a temperature-sensitive large T-antigen, is non-tumorigenic, retains features of transitional urothelium when propagated on serum -free growth medium and is thus, an excellent in-vitro model of human urothelium [+2 and As+3 can cause the malignant transformation of urothelial cells as evidence by colony formation in soft agar and tumor formation in athymic mice [+3 or Cd+2 would alter the expression of E- or N-cadherin in-vitro and in-vivo.The development of urothelial cancer has a strong association with the environment . This lan cancer –22. Studn cancer . This sun cancer . The hig smokers , 26, and smokers , 28. In  smokers . The humothelium . Using tmic mice –32. Thismic mice . These tmic mice . The URO6 cells in the dorsal thoracic midline of athymic nude (NCR-nu/nu) mice. Tumor formation and growth were assessed externally with a ruler on a weekly basis and the maximal tumor size was 2.5 cm. All mice were sacrificed by 10 weeks after injection or when clinical conditions dictated euthanasia . This study adhered to all recommendation dictated in the Guide for the Care and Use of Laboratory Animals of the NIH. The specific protocol was approved by the University of North Dakota Animal Care Committee (IACUC#1110-2C). All efforts were taken in order to minimize animal suffering, and mice were euthanized when clinical condition dictated. Animals were sacrificed by 10 weeks post tumor transplantation by CO2 inhalation and euthanasia conformed to AVMA Guideline on Euthanasia.Mouse heterotransplants of the UROtsa transformed cell cultures were produced by subcutaneous injection at a dose of 1 X 102 tissue culture flasks using Dulbecco’s modified Eagle’s medium (DMEM) containing 5% v/v fetal calf serum in a 37°C, 5% CO2: 95% air atmosphere [+2- transformed lines and the 6 individual isolates of the As+3-transformed UROtsa cells has been described previously [+3 and Cd+2, the UROtsa parental cells were exposed to 1.0 μM NaAsO2 or CdCl2 and passaged in the presence of NaAsO2 or CdCl2 for 30 days. During this exposure period, the ability of the cells to form colonies in soft agar was determined every five passages using protocols described previously [The UROtsa cell line was initially obtained from John Masters formerly of the Institute of Urology, University College London and maintained in 75 cmmosphere . The isoeviously –32. The +3-and Cd+2-transformed cell lines were seeded at a density of 105 cells in T-25 cm2 Ultra-low attachment flasks . The growth medium consisted of a 1:1 mixture of Dulbecco’s modified Eagles’ medium and Hams’s F-12 growth medium supplemented with selenium (5 ng/ml), insulin (5 μg/ml), transferrin (5 μg/ml), hydrocortisone (36 ng/ml), triiodothyronine (4 pg/ml), and epidermal growth factor (10 ng/ml). The cells were allowed to form spheres for 8 days after which time the spheroids were harvested by centrifugation for RNA and protein isolation, and for injection into immune compromised mice. The protocol for the transplantation of the transformed UROtsa cell lines into immune compromised mice has been described previously [For the generation of spheroids, the Aseviously .The preparation of total RNA and protein from cultured cells and tissues have been described previously –32. The The expression of E- and N-cadherin protein was determined by Western blotting using protocols that have been previously published by this laboratory –32. Brie2. Cells at confluent density were then fixed and stained using previously described procedures [For immuno-localization, the cells were grown in 24 well plates containing 12 mm glass coverslips at 37°C, 5% COocedures , 32. ConTM  following the manufacturer’s recommended protocol. The N-cadherin antibody (Life Technology) and the E-cadherin antibody (Santa Cruz Biotechnology) were used at a dilution of 1:100. Liquid diaminobenzidine was used for visualization. Slides were rinsed in distilled water, dehydrated in graded ethanol, cleared in xylene, and coverslipped. Normal human kidney and Dako Universal Negative were used as positive and negative controls.The mouse subcutaneous heterotransplants were fixed in 10% neutral buffered formalin for 16–18 hours. All tissues were transferred to 70% ethanol and dehydrated in 100% ethanol. Dehydrated tissues were cleared in xylene, infiltrated, and embedded in paraffin. Tissue sections were cut at 3–5 μm for use in immuno-histochemical protocols. Prior to immuno-staining, sections were immersed in preheated citrate buffer pH 6.0 and heated in a steamer for 20 minutes. The sections were allowed to cool to room temperature and immersed into TBS-T  for 5 minutes. The immuno-staining was performed using the Dako ARK2 in PBS followed by the addition of 0.5 ml 10 mM EDTA. Non-specific staining was blocked using 1% bovine serum albumin (BSA) for 15 min on ice. Phycoerythrin (PE)-conjugated N-cadherin  was added to cells at 1:10 dilution and incubated at 4°C in the dark for 30 min. Samples were washed two times in 0.1% BSA in PBS before flow cytometry analysis. Cells were counted with a BD LSRII using FACSDiva software. Results were gated to exclude doublets and identify the singlet population.Cells were dissociated in 0.1% trypsin, 0.5 mM CaClAll experiments were performed in triplicate and the results are expressed as the standard error of the mean. Statistical analyses were performed using GraphPad Prism® software using separate variance t-tests, ANOVA with Tukey post-hoc testing. Unless otherwise stated, the level of significance was 0.05.+3-transformed UROtsa cell lines, and the 7 independent isolates of the Cd+2-transformed UROtsa cell lines and analyzed for the expression of N-cadherin mRNA using real-time PCR. The results of this analysis showed a very low expression of N-cadherin mRNA in the parental UROtsa cells . During this period of exposure, the cells did not form colonies in soft agar. This suggests that long term exposure does not induce the expression of N-cadherin and that the actual transformation process may be required to induce the expression of the gene.Total RNA was isolated from confluent cultures of parental UROtsa cells, the 6 independent isolates of Assa cells . In contll lines  and in all lines . The ele protein . Identicll lines  and in all lines . The As++3-transformed UROtsa cell lines, and the 7 independent isolates of the Cd+2-transformed UROtsa cell lines was also used to determine the expression of E-cadherin mRNA using real-time PCR. The results of this analysis showed that the parental UROtsa cell line as well as all the As+3-  to moderately positive for 20–40% of the population  to greater than 60% moderately positive (Cd#5) , three of the 6 isolates had moderate levels of staining with some cells staining intensely  and two isolates had high levels, greater than 85% of the population, with many intensely staining cells   .+3-transformed cell lines, approximately 98% of the cells of the As#6 cell line and 40% of the cells of the As#3 cell line showed strong to moderate expression of N-cadherin respectively. The Cd#1 and Cd#5 cell lines showed moderate expression in about 40 and 60% of the cells respectively . In all 5 cell lines, E-cadherin staining was uniformly intense, localized to the plasma membrane, and expressed by all the cells in the monolayer’s examined for each cell line , a stem cell marker, and N-cadherin mRNA. It was demonstrated that spheroids from all 4 cell lines had an elevated expression of ALDH1A when compared to their parent cell lines . In contTumor transplants were generated from the above 4 spheroids and assessed for their expression of N-cadherin using immuno-histochemistry. It was demonstrated that none of the tumor transplants generated from the spheroids stained for the N-cadherin protein . Higher The expression of N-cadherin and E-cadherin mRNA and protein was determined by real-time PCR and western blotting in 3 commonly utilized urothelial cancer cell lines. It was demonstrated the N-cadherin mRNA  and prot+3 and Cd+2, providing evidence that the transformed cells were undergoing EMT. Evidence that N-cadherin would be elevated in the As+3-and Cd+2-transformed cell lines came from an earlier microarray analysis of 6 independent isolates of the As+3-transformed cell lines and 5 independent isolates of the Cd+2-transformed cell lines [+3-and Cd+2-transformed cells were isolated to determine the degree of heterogeneity in gene expression that might occur among isolates malignantly transformed using identical conditions of exposure to the toxicant. The analysis of the expression patterns of these cell lines using the Affymetrix 133 plus 2.0 chip showed that N-cadherin was the most induced gene in the As+3-transformed cell lines (56.9 fold) and the 5th most induced gene in the Cd+2-transformed cell lines (34.9 fold). Based on this earlier data it was expected that each individual cell line would demonstrate an increase in the expression of N-cadherin mRNA and protein. As expected, an increase in overall N-cadherin mRNA and protein was confirmed by real-time PCR and western blotting for each cell line. However, immuno-localization studies on N-cadherin showed a marked heterogeneity within each cell line regarding the % of cells that were leading to the increased N-cadherin expression noted by real-time PCR and western blotting. This was especially noteworthy for two of the seven Cd+2-transformed cell lines and one of the six As+3-transformed cell lines where less than approximately 5% of the cells showed reactivity for N-cadherin while four of the Cd+2-and three of the As+3-transformed cell lines demonstrated moderate to strong N-cadherin reactivity between 20 and 50% of the cells. However, the transformed cell lines with the highest expression of N-cadherin via Western analyses, As#5, As#6, and Cd#5 had not only cells stained moderately to strongly for N-cadherin at the plasma membrane but also had between 60–90% of the population positive for N-cadherin. Thus, these results suggest that the expression level of N-cadherin by real-time PCR and western analysis for the overall culture is not necessarily indicative of the expression levels of the individual cells in the culture. This has several implications on the interpretation of results from individual cell cultures displaying this degree of heterogeneity. One is that the level of N-cadherin expression in the cells that do express N-cadherin is often much higher than would be expected from real-time PCR and western blotting of the entire culture. This is especially true for the cell lines where less than 15% of the cells are expressing N-cadherin. This is important since it does support the concept that this minority population of cells is undergoing the process of EMT and possibly cadherin switching. The second implication is more practical and it is the limitation that such heterogeneity within an individual culture places on the use of such a culture system for mechanistic studies. In the present case, mechanistic studies on the regulation of N-cadherin could be very misleading since, in many of the cell lines, the majority of the cells do not express N-cadherin even though N-cadherin mRNA and protein can be determined for each line. This is especially noteworthy in the present study since a microarray analysis of the cell lines indicated that N-cadherin was one of the most highly induced genes and the cell cultures studies using real-time PCR and western blotting were supportive of the finding of induced expression.The initial goal of this study was to show that N-cadherin expression was increased in UROtsa cells malignantly transformed by Asll lines . The mul+3-and Cd+2-transformed cell lines is expected as a hallmark of malignant transformation. However, the finding of variation of N-cadherin expression within the individual cells comprising each cell line was not expected. The finding that N-cadherin expression was variable among the cells comprising each cell line appears to be unique and not a reflection of a cell culture system or a series of cell lines that are composed of a variety of cells with diverse expression profiles. The expression of E-cadherin was determined in the present study and expression was uniform within all the cell lines and among the individual cell lines. There were only very rare profiles of cells from any of the cell lines that did not express E-cadherin. Co-localization was performed for N-cadherin and E-cadherin in the As#6 cell line where the majority of cells express N-cadherin and all the cells that expressed N-cadherin also expressed E-cadherin. This finding also suggested that while N-cadherin expression was elevated, there was no evidence of “cadherin switching” since there was no noticeable reduction in E-cadherin expression. To assure that the reagents and methods utilized accurately detected E- and N-cadherin, their expression was also determined in 3 commonly used human urothelial cancer cell lines. The results showed that those cell lines know to have mesenchymal character showed elevated expression of N-cadherin and only background expression of E-cadherin and vice versa. Other studies on the UROtsa cell lines have shown a uniform expression of proteins within the cells of each line; SPARC in the parental UROtsa cells [+3-and Cd+2-transformed cell line is composed of both cells positive and negative for the expression of N-cadherin is that each culture is a snap shot in time as each cell line progresses to one containing a more homogeneous population of cells expressing N-cadherin. This might also suggest that cells transformed by As+3 have a greater tendency for progression since N-cadherin was more pronounced in the As+3-transformed cell lines.The finding of the heterogeneity in gene expression among the individual independent isolates of the Assa cells , and enosa cells , 34–38. sa cells . EMT mig+3-transformed cells and the 7 isolates of Cd+2-transformed cells showed no staining for the N-cadherin protein when examined by immune-histochemistry. Multiple slides from each tumor were examined to rule out the possibility of areas of focal staining for N-cadherin. Sections of human kidney were included as a control and showed the staining pattern known for N-cadherin. An examination of N-cadherin protein expression in tumor lysates by Western blotting also showed only background staining for N-cadherin. The finding that the tumor cells comprising the tumor transplants had no expression of N-cadherin was somewhat surprising since all the cell lines showed some expression of N-cadherin and the As#6 cell line showed the majority of cells as staining positive for N-cadherin on both immune-fluorescence examination and by flow cytometry. In contrast, all the tumors from all the cell lines stained intensely and uniformly for E-cadherin. One explanation for this finding would be that the cells expressing N-cadherin are not able to establish (seed) a tumor in the immune compromised mice following subcutaneous injection. This explanation would be in agreement with the classical view of EMT. In this view, it is widely accepted that the process of EMT is associated with a cell’s ability to escape the basement membrane and disseminate into the lymph nodes, blood stream or local body cavity [+3-and Cd+2-transformed cells would be consistent with E- and not N-cadherin expressing cells “seeding” a site for tumor growth. For this explanation to be viable, the As+3- and Cd+2- transformed cell lines should possess a subpopulation of cells capable of initiating tumor formation, or what is commonly called a population of cancer-initiating cells (CICs) [+3- and Cd+2- transformed cell lines as a putative subpopulation of cells (CICs) that would be able to establish tumors in immune compromised mice. It was demonstrated that these CICs had only background levels of N-cadherin mRNA, elevated levels of ALDH1 mRNA and were able to establish tumors in immune compromised mice. The tumors produced by the injection of these CICs subcutaneously showed no staining for the N-cadherin protein by immuno-histochemical examination and the tumors displayed histologies indistinguishable from those produced by injection of the original cell lines. This is especially noteworthy for the CICs isolated from the As#6 cell line since it was composed almost entirely of cells that expressed N-cadherin. These findings provide strong supporting evidence that the cells that expressed N-cadherin in the As+3- and Cd+2-transformed cell lines were unable to “seed” tumors in the immune compromised mice. An alternate explanation would be that the CICs simply lose N-cadherin expression when they are unable to attach to the surface of the cell culture flask. However, this explanation is less likely since it would be surprising that these cells would not regain expression of N-cadherin once the cells comprised the tumor mass. The As+3 and Cd+2 tumor transplants did not grow back when put into cell culture, therefore it is not known if they would regain the ability to express N-cadherin when grown in a serum containing environment. The expression of E and N-cadherin in the tumors produced by the commercially available cell lines was also determined and it was found that the expression of E-cadherin was moderate to high, whereas the expression of N-cadherin was weak in the tumors produced by the J82 cell line and was absent in the tumors produced by the RT4 cell line. Thus this data recapitulates the results of the tumors produced by the As+3-and the Cd+2-transformed cell lines.The tumor transplants produced from the 6 isolates of Asy cavity –42. On ty cavity –42. In ts (CICs) –45. ThisThe results also serve as a caution for laboratories using cell culture models of human cancer, especially when they are used to produce tumor transplants. The results of the present study suggest that the population of cultured cells producing a tumor transplant may be very rare and distinct from the expression pattern of the overall culture.in-vitro, however tumors formed by the subcutaneous injection of these transformed cells do not express N-cadherin. This finding is in agreement with the classical view of EMT where it is thought that the expression of N-cadherin is associated with the dissemination of the tumor, whereas the expression of E-cadherin is associated with tumor seeding. In addition, the study also shows that a small proportion of cells present in the culture are undergoing the process of EMT and possibly cadherin switching.Urothelial cells transformed by the environmental toxicants arsenite and cadmium express N-cadherin S1 Fig(A-D). Expression of N-cadherin protein in tumor transplants generated from As#3, As#6, Cd#1 and Cd#5 cell lines respectively. (E-H) Expression of E-cadherin protein in tumor transplants generated from As#3, As#6, Cd#1 and Cd#5 cell lines respectively. The brown color indicates the presence of the protein whereas the blue color indicates the nuclei that were stained with the counterstain hematoxylin. All images are at a magnification of X400. Scale bar = 50 μm and is shown for panels A-H.(TIF)Click here for additional data file.S2 FigN-cadherin (green) was found to be localized to the plasma membrane, when present, in the parent and transformed UROtsa cell line:  UROtsa parent and  As#6. Co-localization (yellow) of N-cadherin (green) and E-cadherin (red) is also shown . High magnifications of the selected pictures are shown in the panels to the right . The regions magnified are indicated by the white boxes on the lower magnification images. All scale bars = 10 μm.(TIF)Click here for additional data file.S3 Fig(A-D). Expression of N-cadherin protein in tumor transplants generated from As#3, As#6, Cd#1 and Cd#5 cancer initiating cell spheroids respectively. (E). Human kidney stained for N-cadherin as a positive control. The brown color indicates the presence of the protein whereas the blue color indicates the nuclei that were stained with the counterstain hematoxylin. All images are at a magnification of X400. Scale bar = 50 μm and is shown for panels A-E.(TIF)Click here for additional data file.S4 FigThe brown color indicates the presence of the protein whereas the blue color indicates the nuclei that were stained with the counterstain hematoxylin. All images are at a magnification of X400. Scale bar = 50 μm.(TIF)Click here for additional data file.


Skin toxicity is the most common toxicity caused by Epidermal Growth Factor Receptor (EGFR) inhibitors, and has been associated with clinical efficacy. As EGFR inhibitors enhance the expression of antigen presenting molecules in affected skin keratinocytes, they may concurrently facilitate neo-antigen presentation in lung cancer tumor cells contributing to anti-tumor immunity. Here, we investigated the modulatory effect of the EGFR inhibitor, erlotinib on antigen presenting molecules and PD-L1, prominent immune checkpoint protein, of skin keratinocytes and lung cancer cell lines to delineate the link between EGFR signaling pathway inhibition and potential anti-tumor immunity. Erlotinib up-regulated MHC-I and MHC-II proteins on IFNγ treated keratinocytes but abrogated IFNγ-induced expression of PD-L1, suggesting the potential role of infiltrating autoreactive T cells in the damage of keratinocytes in affected skin. Interestingly, the surface expression of MHC-I, MHC-II, and PD-L1 was up-regulated in response to IFNγ more often in lung cancer cell lines sensitive to erlotinib, but only expression of PD-L1 was inhibited by erlotinib. Further, erlotinib significantly increased T cell mediated cytotoxicity on lung cancer cells. Lastly, the analysis of gene expression dataset of 186 lung cancer cell lines from Cancer Cell Line Encyclopedia demonstrated that overexpression of PD-L1 was associated with sensitivity to erlotinib and higher expression of genes related to antigen presenting pathways and IFNγ signaling pathway. Our findings suggest that the EGFR inhibitors can facilitate anti-tumor adaptive immune responses by breaking tolerance especially in EGFR driven lung cancer that are associated with overexpression of PD-L1 and genes related to antigen presentation and inflammation. Lung cancer remains a leading cause of cancer death in the Unites States, with 158,040 estimated death to occur in 2015 x100.H441, HLA-A2http://www.broadinstitute.org/ccle/home. Lung cancer cell lines were divided into two groups, PD-L1high and PD-L1low lung cancer cell lines, according to median log2 Robust Multi-array Average (RMA) values. Log2 RMA values were used to evaluate selected gene expression between PD-L1high and PD-L1low lung cancer cell lines.The gene expression dataset of 186 lung cancer cell lines and drug sensitivity data of 91 lung cancer cell lines were obtained at the following site : http://2 RMA values of selected gene expression between PD-L1high and PD-L1low lung cancer cell lines, T cell mediated cytotoxicity on lung cancer cell lines treated with or without erlotinib, and surface expression of MHC-I, MHC-II, and PD-L1 of various cell lines treated with IFNγ and/or erlotinib Non-parametric Wilcoxon test was used for paired analysis to evaluate the impact of EGFR inhibitors on surface expression of MHC-I, MHC-II, and PD-L1 proteins between IFNγ or Erlotinib treated or untreated lung cancer cell lines sensitive or resistant to erlotinib.All statistical analysis was performed using Prism version 6.0 . Wilcoxon-Mann-Whitney test was used to compare LogIn order to elucidate the immune-mechanism of skin rash caused by EGFR inhibitors, we first investigated the modulatory effects of erlotinib, one of EGFR inhibitors, on the expression pattern of antigen presenting molecules and PD-L1 on skin keratinocytes. Previously, one of malignant skin keratinocyte cell line, A431, was used to demonstrate the synergistic effects of EGFR inhibitors on the expression of MHC-I and MHC-II because this particular cell line expresses abnormally high level of EGFR compared to normal skin keratinocytes, thus the effect of EGFR inhibition would be accentuated ,36,37. HAs expected, HaCaT cells up-regulated the surface expression of both MHC-I and MHC-II with IFNγ treatment. However, only the expression of MHC-I on HaCaT cells was slightly increased by the treatment with EGFR inhibitor, Erlotinib. Both treatments did not alter the surface expression of EGFR on HaCaT cells . Next, wPollack at al has previously reported that EGFR inhibitors synergistically up-regulated the expression of antigen presenting proteins in one of squamous cell carcinoma (SCC) of head and neck cancer cell lines among other cell lines originated from various cancer such as melanoma or colon cancer . To our 50 <8) compared to those resistant to erlotinib (EC50 ≥8), but it was not statistically significant likely due to the heterogeneity of lung cancer cell lines and small number of lung cancer cell lines tested  specific for MART-1 (M27) peptide restricted to HLA-A2 as effector T cells . HLA-A2+nhibitor . H441 luhttp://www.broadinstitute.org/ccle) in relation to the PD-L1 expression. The gene expression profiles of 186 lung cancer cell lines were retrieved from CCLE database, and 186 lung cancer cell lines were divided into two groups, PD-L1high and PD-L1low according to the median expression value of PD-L1  and class II antigen presenting pathway  , and chemokine receptor such as CXCR1 (IL-8 receptor) while PD-L1low lung cancer cell lines were associated with higher expression of CXCR4 and CXCR7 that are essential for lung cancer progression and angiogenesis  , and infnd CD74)  comparedfindings . Interess Tables  38,39].,39.high Human keratinocytes are an essential component in the skin immune system as they can regulate both innate and adaptive immune responses through the production of various cytokines, chemokines, and anti-microbial peptides in response to skin injury, inflammation, and infections ,41. In ain vitro sensitivity to erlotinib, one of the three EGFR inhibitors that have been approved for the first line therapy of EGFR-mutated lung cancer.As the clinical benefit derived from the treatment with EGFR inhibitors is associated with the severity of the skin toxicity in lung and colorectal cancer, we hypothesized that in addition to the well-known cytotoxic effects of EGFR inhibition, a similar modulation of antigen presenting molecules and PD-L1 proteins by EGFR inhibition in tumor cells may be implicated in augmenting anti-tumor immune responses, thus contributing to an additional mechanism of anti-tumor activity in these cancers. Therefore, we investigated the impact of EGFR inhibition on the expression of antigen presenting molecules and PD-L1 proteins in lung cancer cell lines with a wide range of 50 <8) and those resistant to erlotinib (EC50 ≥8). There was a trend toward higher baseline surface expression of PD-L1 and MHC-I proteins in lung cancer cell lines sensitive to Erlotinib, and significantly increased expression of MHC-I, MHC-II, and PD-L1 in response to IFNγ in lung cancer cell lines sensitive to erlotinib when compared to those resistant to erlotinib. This is an interesting and important finding, suggesting that there might be a crosstalk between the EGFR and IFNγ receptor mediated signaling pathways that are essential in the transcriptional control of immune-related genes especially for classical antigen presenting pathway involving MHC-I and MHC-II.First, we observed a distinct difference in the expression pattern of antigen presenting molecules and PD-L1 between lung cancer cell lines sensitive to erlotinib (EChigh lung cancer cell lines display a pro-inflammatory phenotype with great potential for neo-antigen presentation and recruiting immune cells that are essential to initiate anti-tumor immunity. As the overexpression of PD-L1 in EGFR-driven lung cancer cells may be a mechanism of immune-tolerance to evade the tumor killing by infiltrating immune cells, the PD-1 and PD-L1 axis can be a reasonable therapeutic target to break the immune-tolerance and to enhance anti-tumor immunity in EGFR-driven lung cancer.Overexpression of PD-L1 has been implicated as a mechanism of immune-evasion in lung cancer as well as other solid tumors, and has been reported to be associated with the presence of sensitizing EGFR mutations in selected lung cancer cell lines –23 and rBlockade of PD-1/PD-L1 axis is the novel immunotherapeutic modality with a striking clinical efficacy in the treatment of many solid tumors such as melanoma, renal cell carcinoma, non-small cell lung cancer, and certain malignancies with mismatch-repair deficiency –29. Antihigh CD8+ T cells and their subsequent expansion upon PD-1/PD-L1 blockade predicts the effective tumor regression, suggesting the direct role of anti-tumor T cell responses in immunotherapy strategies that target the PD-1/PD-L1 axis [The presence of intratumoral infiltrating PD-1-L1 axis ,52. EGFR-L1 axis ,22. HereIn summary, we seek to elucidate a novel immune-mechanism of EGFR inhibitor-induced skin rash and corresponding anti-tumor immunity in lung cancer. First, we demonstrated that PD-L1 overexpression in lung cancer is associated with increased sensitivity to EGFR inhibitors, and a higher expression of EGFR and genes critical to neo-antigen presentation, and that down-modulation of PD-L1 may be an additional mechanism of anti-tumor activity of EGFR inhibitors in augmenting anti-tumor T cell responses. Thus, our study provides a rationale for conducting clinical studies to investigate combination therapy with EGFR inhibitors and PD1/PD-L1 axis blocking antibodies especially in EGFR-driven lung cancer with either as a result of sensitizing mutations or overexpression of wild type EGFR.


Aberrant expression of the RON receptor tyrosine kinase, a member of the MET proto-oncogene family, in breast cancer and non-small cell lung cancer (NSCLC) has therapeutic implication. Here we evaluated the efficacy of a novel anti-RON antibody-drug maytansinoid conjugate Zt/g4-DM1 for treatment of breast and NSCLC xenograft tumors in mouse models and explored a treatment strategy by combination of Zt/g4-DM1 with chemotherapeutics to achieve the maximal therapeutic activity.Mouse monoclonal antibody Zt/g4 (IgG1a/κ) specific to human RON was conjugated to DM1 via thioether linkage to form Zt/g4-DM1 with a drug-antibody ratio of 4:1. Several breast cancer and NSCLC cell lines, expressing different levels of RON, were used as the model. Immunofluorescence was used to determine Zt/g4-induced RON internalization. Flow cytometric analysis and cell viability assay were used to determine the effect of Zt-g4-DM1 on cell cycle and death. Mouse xenograft NSCLC models were used in vivo to determine the therapeutic efficacy of Zt/g4-DM1 alone or in combination with chemotherapeutics.In vitro, Zt/g4 treatment of breast cancer and NSCLC cells rapidly induced cell surface RON internalization, which results in intracellular delivery of DM1 sufficient to arrest cell cycle at G2/M phase, reduce cell viability, and cause massive cell death. In mouse tumor xenograft models, Zt/g4-DM1 at 20 mg/kg in a Q12 × 2 regimen effectively blocked breast cancer and NSCLC cell- mediated tumor growth. More than 95 % inhibition of tumor growth among three tumor xenograft models tested was achieved according to the measured tumor volume. The minimal dose to balance the tumor growth and inhibition (tumoristatic concentration) was established at 2.02 mg/kg for H2228, 1.94 mg/kg for H358 cell, and 6.25 mg/kg for T-47D cell-mediated xenograft tumors.Zt/g4 is highly effective in RON-directed drug delivery for targeted inhibition of NSCLC cell-derived tumor growth in mouse xenograft models. This work provides the basis for clinical development of humanized Zt/g4-DM1 for potential cancer therapy in the future. Thus, ADCs represent a promising therapeutic modality for the clinical management of cancer.Antibody-directed drug delivery in the form of antibody-drug conjugates (ADC) is a promising anti-cancer strategy –3. Strucwww.clinicaltrials.gov). Evidence accumulated from various studies showed that inhibition of RON has the therapeutic effects on cancer cell growth, migration, and survival [The RON receptor tyrosine kinase is a member of the MET proto-oncogene family implicated in the pathogenesis of BC and non-small cell lung cancer (NSCLC) –20. Immusurvival –29. Howesurvival –29. The survival . Thus, iFor the last several years, we have focused on anti-RON mAb-directed drug delivery for targeted cancer therapy , 30–32. Human BC cell lines MCF-7, DU4475, MDA-MB-231, T-47D, and NSCLC cell lines H1993, H2228, and H358 were from ATCC . Mouse anti-RON mAbs Zt/g4, Zt/c1 and rabbit anti-RON IgG antibodies were as previously described . Goat anZt/g4 was conjugated with DM1 at a drug-antibody ratio of 4:1 by through the linker SMCC as previously described , 35. Con6 cells per ml in PBS) were treated with Zt/g4 at saturating concentrations followed by incubation in parallel with the QIFIKIT® beads and goat F(ab’)2 F0479. After establishing a calibration curve, the number of RON receptor on the cell surface was determined by interpolation following the manufacturer’s instruction.Cell surface RON was quantitatively determined by the immunofluorescence assay using QIFKIT® reagents from DAKO . Briefly, cells (1 × 106 cells per dish) were treated with 5 μg/ml of Zt/g4-DM1 at 37 °C for 24 h. Cells were then labeled with propidium iodide followed by Accuri flow cytometric analysis. Cell cycle changes were determined by measuring DNA contents as previously described [BC and NSCLC cells (1 × 10escribed .Cellular proteins (50 μg per sample) were separated in an 8 % SDS- PAGE under reduced conditions. Cellular proteins such as PARP were detected in Western blotting using specific antibodies. Membranes also were reprobed with anti-actin antibody to ensure equal sample loading .5 cells per well in a 6-well plate were treated with 5 μg/ml of Zt/g4-DM1 for various times followed by goat anti-mouse IgG coupled with FITC [BC and NSCLC cells at 1 × 10ith FITC . Nuclearith FITC . CellulaCell viability 96 h after Zt/g4-DM1 treatment was determined by the MTT assay , 36. Per6 cancer cells in the subcutaneous space of the right flank as previously described [3. To evaluate the Zt/g4-DM1 efficacy, mice were injected with Zt/g4- DM1 at 20 mg/kg in a Q12 × 2 regimen. Tumor growth was observed up to 52 days. In study of Zt/g4-DM1 in combination with gemcitabine, tumor-bearing mice were treated with gemcitabine alone at 60 mg/kg in a Q4 × 4 schedule [3 or if tumors became necrotic or ulcerated through the skin.All experiments on mice were approved by the TTUHSC Institutional Animal Care Committee. Female athymic nude mice at 6 weeks of age  were injected with 5 × 10escribed , 33. Micschedule , Zt/g4-Dschedule , 25. Anit test. Chi-squared analysis was used for correlational study. Isobolograms were used for analysis of synergism in drug combination studies. Statistical differences at p <0.05 were considered significant.GraphPad 6 software was used for statistical analysis. Results are shown as mean ± SD. The data between control and experimental groups were compared using Student To study the effect of Zt/g4 on RON internalization, we first determined the number of RON molecules expressed on cell surface using the QIFKIT® fluorescence-based quantitative method Fig. . The cal50). The calculated IE50 values were >100 h for MCF-7, 14.32 h for DU4475, 11.71 h for MDA- MB-231, 23.46 h for T-47D, 11.65 h for H1993, 7.47 h for H358, and 9.84 h for H2228 cells  and the number of RON receptors per cell is shown in Fig. 50 values is correlated with the number of RON molecules expressed on the cell surface. It appeared that the minimal number of cell-surface RON molecules required for Zt/g4-DM1 to achieve a 95 % reduction in cell viability (EC95) is around 10,000 molecules per cell. A decrease in the level of RON receptors per cell correlated proportionally with the diminished efficacy of Zt/g4-DM1. Thus, cancer cells expressing ~10,000 RON molecules on cell surface per cell appears to be required for Zt/g4-DM1 in vitro to show the maximal activity.A comparison between the efficacy of Zt/g4-DM1 . The TSC is a dose that reaches a balance in vivo between the tumor growth and inhibition. To this end, mouse xenograft tumor models of H1993, H288, H358, and T-47D cells were established. Xenograft models for MDA-MB-231 and DU4475 cells were unable to be established. Zt/g4-DM1 at 20 mg/kg with a Q12 × 2 schedule was used for in vivo studies, which was designed according to our previous studies and the terminal half-life (t½: 6.01 days) of Zt/g4-DM1 (25). As shown in Fig. A comparison of tumor weight between the control and Zt-g4-DM1 treated group at the end of experiments was shown in Fig. We calculated the TSCs based on the terminal half-life of Zt/g4-DM1 in relationship with the tumor regrowth curve Fig. . Zt/g4-DTo exclude any possibilities that reduction of tumor is related to general toxicity of Zt/g4- DM1 in vivo, we monitored body weight of individual tumor-bearing mice before and after treatment Fig. . All micWe first studied the activities of Zt/g4-DM1 in combination with chemotherapeutics. BC and NSCLC cell lines were treated for 96 h in vitro with different amounts of gemcitabine, Zt/g4-DM1 or their combinations at 1 to 1 molar ratio. Results in Fig. p: <0.05) was 88.43 % for H358 and 90.62 % for T-47D. Measurement of tumor weight at the end of the study further confirmed an increase in inhibition of tumor growth. For H358 xenograft tumors, the average tumor weight from the combined treatment groups (0.24 g ± 0.17) was significantly less than that from the Zt/g4-DM1-treated group  and from the gemcitabine-treated group  . Thus, Zt/g4-DM1 in combination with gemcitabine results in the increase in inhibition of T-47D and H358 xenograft tumors.We then performed the combination therapy in mouse xenograft tumors, in which Zt/g4- DM1 at 10 mg/kg in the Q8 × 2 regimen was combined with gemcitabine at 60 mg/kg in the Q4 × 4 schedule Fig. . Gemcita05) Fig. . Similar50 values for cell viability and the levels of RON expression by cancer cells. Studies from mouse xenograft tumor models confirmed that Zt/g4 is suitable for DM1 delivery in vivo and Zt/g4-DM1 at 20 mg/kg in the Q12 × 2 regimen inhibits tumor growth with a long-lasting effect. The combinational therapy further demonstrated that Zt/g4-DM1 together with chemoagents results further in delaying BC and NSCLC xenograft tumor growth. Thus, Zt/g4-DM1 is effective in the model of anti-RON ADC for treatment of BC and NSCLC overexpressing the RON receptor tyrosine kinase.The study presented here shows the effectiveness of Zt/g4 in RON-targeted DM1 delivery for treatment of BC and NSCLC. Both in vitro and in vivo studies demonstrated the capability of Zt/g4 in induction of RON internalization by cancer cells. Also, Zt/g4-directed delivery of DM1 is sufficient to cause cell cycle arrest in G2/M phase, to inhibit cell proliferation and to cause massive cell death. These functional analyses help to establish a correlation between the IC50 values for BC cell lines expressing the high levels of RON (more than 8000 RON molecules per cell) is in the range of 10 ~ 16 h. In contrast, the IE50 values for NSCLC H2228 and H358 cell lines expressing similar levels of RON were at the 7 to 10 h ranges. The mechanism(s) underlying such differences currently is unknown. Regardless of the difference, Zt/g4-induced RON internalization is highly effective with more than 85 % of cell surface RON internalized within 36 h. In the case of H358 cells expressing ~15,000 RON molecules per cell, it translates into 12,750 RON receptors that are internalized within 36 h. This is equivalent to 51,000 DM1 molecules delivered into a single cell, which is sufficient to cause cell cycle arrest. In cell lines such as H1993 expressing the low level of RON, Zt/g4 was able to induce RON internalization with IE50 similar to those of other cancer cell lines (Fig. Selection of Zt/g4 for DM1 conjugation and delivery is based on its unique feature in induction of RON internalization , 34. We nes Fig. . However50 values ranging from 2 to 6 μg/ml. Analysis of cellular markers for apoptotic death further confirmed that Zt/g4-DM1 treatment activates PARP as evident by the presence of the PARP fragment. These findings indicate that DM1-mediated cell cycle arrest leads to initiation of an intracellular apoptotic pathway. Finally, we discovered that the efficacy of Zt/g4-DM1 in vitro directly correlates with the levels of the RON expression by cancer cells. For Zt/g4-DM1 to achieve a significant reduction in cell viability, the minimal RON molecule expressed by cancer cells should be at ~8000 receptors per cells. We reason that such a correlational relationship is useful for predicting the in vitro effectiveness of Zt/g4-DM1 against cancer cells, which helps to select potential target cancer cells for evaluation in vivo.The action of DM1 delivered by Zt/g4 was displayed in both BC and NSCLC cells. First, we showed by flow cytometric analysis that the Zt/g4-DM1 internalization causes cell cycle arrest in G2/M phase, which is a feature of DM1 that impairs microtubule dynamics . This efResults from mouse xenograft tumor models demonstrate the efficacy of Zt/g4-DM1 in inhibition of tumor growth. It is possible that Zt/g4-mediated immunological activities may also contribute the observed therapeutic efficacy. As described in other ADCs, antibody-dependent cell-mediated cytotoxicity, antibody dependent cellular phagocytosis, and complement- dependent cytotoxicity play a role in vivo in the ADC anticancer activity . We usedStudies using H358 and T-47D xenograft models demonstrated the increase in therapeutic activity between Zt/g4-DM1 and gemcitabine in inhibition of tumor growth. We showed from in vitro studies that the combination of Zt/g4-DM1 with chemotherapeutics at the same molar ratio further reduce viability of BC and NSCLC cells. Analysis of the average tumor weight at the end of the study also confirmed the increase in therapeutic activity between Zt/g4-DM1 and chemotherapeutics. This indicates that the inhibition can be synergized through different mechanisms of action. It is worth to note that the synergism of Zt/g4-DM1 with gemcitabine is dependent on individual cancer cell lines in the particular dose range. We reasoned from the combination therapy that since BC and NSCLC tumors are highly malignant at the later stage, Zt/g4-DM1 in combination with chemotherapeutics could be a strategy for targeted cancer therapy.We present evidence in this study that anti-RON antibody directed delivery of the highly potent drug DM1 in the form of ADC is a therapeutic strategy for targeted treatment of breast and NSCLC cancer in animal models. Results from various in vitro and in vivo experiments showed that Zt/g4-DM1 not only significantly decreases viability of breast cancer and NSCLC cells but also dramatically delays the tumor growth in mouse xenograft tumor models. Moreover, we demonstrated that Zt/g4-DM1 in combination gemcitabine significantly increases the inhibitory effect on xenograft tumor growth. These findings lay the foundation for clinical development of anti-RON ADC Zt/g4-DM1 for potential cancer therapy.


The aim of this paper was to examine whether cell-penetrating peptides (CPPs) such as transportan 10 (TP10) or protein transduction domain (PTD4) may improve the anticancer activity of cisplatin (cPt). The complexes of TP10 or PTD4 with cPt were used in the experiments. They were carried out on two non-cancer (HEK293 (human embryonic kidney) and HEL299 (human embryo lung)) and two cancer  and OS143B (human osteosarcoma 143B)) cell lines. Both complexes were tested (MTT assay) with respect to their anticancer or cytotoxic actions. TAMRA (fluorescent dye)-stained preparations were visualized in a fluorescence microscope. The long-term effect of TP10 + cPt and its components on non-cancer and cancer cell lines was observed in inverted phase contrast microscopy. In the MTT test (cell viability assay), the complex of TP10 + cPt produced a more potent effect on the cancer cell lines  in comparison to that observed after separate treatment with TP10 or cPt. At the same time, the action of the complex and its components was rather small on non-cancer cell lines. On the other hand, a complex of another CPP with cPt, i.e., PTD4 + cPt, was without a significant effect on the cancer cell line (OS143B). The images of the fluorescent microscopy showed TAMRA-TP10 or TAMRA-TP10 + cPt in the interior of the HeLa cells. In the case of TAMRA-PTD4 or TAMRA-PTD4 + cPt, only the first compound was found inside the cancer cell line. In contrast, none of the tested compounds gained access to the interior of the non-cancer cells . Long-term incubation with the TP10 + cPt (estimated by inverted phase contrast microscopy) lead to an enhanced action of the complex on cell viability (decrease in the number of cells and change in their morphology) as compared with that produced by each single agent. With regard to the tested CPPs, only TP10 improved the anticancer activity of cisplatin if both compounds were used in the form of a complex. Additionally, the complex was relatively safe for non-cancer cells. What is more, TP10 also produced an anticancer effect on HeLa and OS143B cell lines. Efficient delivery of therapeutic molecules to cells is a great challenge in modern medicine and pharmacology. Recently, cell-penetrating peptides (CPPs) have received great attention as efficient cellular delivery vectors due to their intrinsic ability to enter cells and mediate uptake of a wide range of macro- or nanomolecular cargos. Generally, CPPs are relatively short cationic peptides which are classified into two groups based on their physicochemical characteristics: amphiphatic and nonamphiphatic. Among the amphiphatic are transportans (TPs) with transportan 10 (TP10) being the one of the most widely explored. CPPs are associated with cargos via covalent bonds or through non-covalent interactions. A large range of chemical agents can be regarded as cargos, i.e., plasmids, DNA, siRNA, proteins, peptides, low molecular weight drugs, and nanoparticles.The mechanisms by which CPPs are transclocated across the biological membranes still remain unclear. It is known, however, that they involve rather an energy-independent cellular process in which different endocytotic and non-endocytotic routes are used. Which of them a CPP will utilize depends on many factors, including, among others, the cargo and the cell type it will enter , being on one hand highly effective in treatment of versatile solid tumors, shows on the other severe toxicity which is a principle factor that limits its usage in clinical oncology. These limitations have motivated an extensive research effort towards development of new strategies for improving platinum therapy. One of them was introduction on the pharmaceutical market two less toxic derivatives of cisplatin, such as carboplatin and oxaliplatin. Nevertheless, its safety profile has not been satisfactory. Recently, new nanotechnology has been employed which includes among others nanocarriers for delivery of platin compounds  and protein transduction domain (PTD4) because they fulfilled mostly the assumed criteria   was attached to the ε-amino function group of Lys7 residue (in case of [Lys7(Prop)]TP10) or the N-terminal amino group (in case of Prop-PTD4) with the use of a 10-fold molar excess of propiolic anhydride. Peptides were cleaved from resins with TFA/phenol/TIPS/water (88:5:2:5) mixture, and this process lasted for 2 h. Next, crude peptides were purified and analyzed with the use of RP-HPLC, and finally characterized by MALDI-TOF mass spectrometry.All peptides Table  were syn3-N3 was synthesized in DCM/DMF solution  using HATU with addition of DIPEA in DCM solution. The final product was lyophilized and purified. Next, it was analyzed with the use of RP-HPLC, and characterized by MALDI-TOF mass spectrometry.TAMRA-PEG7(C(O)-Tra-PEG3-TAMRA)]TP10 and TAMRA-PEG3-Tra-C(O)-PTD4, were obtained with the use of “click chemistry”—a specific 1,3 dipolar Huisgen cycloaddition reaction (Colombo and Peretto 3-N3 with [Lys7(Prop)]TP10 or Prop-PTD4 was carried out in water/tert-butanol medium in the presence of sodium ascorbate and CuSO4 as catalysts. The mixture was stirred at room temperature for 24 h. After the 1,2,3-triazole forming reaction had been completed, crude products were purified by RP-HPLC and characterized by analytical RP-HPLC and MALDI-TOF mass spectrometry.The TAMRA labeled TP10 and PTD4 peptides,  assay involved the following groups of experiments:The compounds were investigated at the concentrations ranging from 0.1–40 μM on all tested cell lines after 24 h of incubation. The aim of this experiment was to find out the non-toxic concentrations in all cell lines.carried out on four tested cell lines: non-cancer HEK293, HEL299 and cancer HeLa, OS143B2)cPt, TAMRA-TP10, TAMRA-TP10 + cPtcarried out on two tested cell lines: non-cancer HEL299 and cancer OS143B3)cPt, TP10, TP10 + cPt, TAMRA4)cPt, TAMRA-PTD4, TAMRA-PTD + cPt, PTD4, TAMRA-PTD4, TAMRANext, only non-toxic concentration of 1 μM was tested after 24, 48, and 72 h of incubation and the further groups were:4 cells/well in a 96-well plate in 100 μl of culture medium and allowed to grow for 24 h before adding cPt, TAMRA, TP10, TP10 + cPt, TAMRA-TP10, TAMRA-TP10 + cPt and PTD4, PTD4 + cPt, TAMRA-PTD4, and TAMRA-PTD4 + cPt. Apart from the first experimental group, the abovementioned agents were only used at the final concentration of 1 μM. After treatment for 24, 48, and 72 h, the cells were washed twice in PBS and then 100 μl of 0.5 mg/ml of MTT in serum-free medium was added into each well. The incubation was continued for a further 3 h (time needed for MTT to become metabolized) at 37 °C. Formazan, a product of the former reaction, was dissolved in 100 μl of acidified isopropanol, and absorbance was measured at 570 nm using a DigiScan 340 microplate reader .MTT assay was based on the protocol described by Park et al. . MTT .The ability of penetration of TP10 and PTD4 was possible to estimate due to their chemical binding with the orange fluorescent dye—TAMRA.5 per 22 mm well (12-well plate) and left for 24 h. Thereafter, they were washed three times in PBS and then 200 μl of DMEM without FBS was added (cells starvation) and left for a further hour. Next, TAMRA-TP10, TAMRA-PTD4, TAMRA-TP10 + cPt, and TAMRA-PTD4 + cPt at a concentration of 10−6 M each were administered to the wells and the samples were incubated for 1 h.The procedure of preparing fluorescent preparations was as follows. Coverslips (∅20 mm) after washing with concentrated HCl and rinsing with 70 % ethanol were placed at the bottom of the 12-well plate. Cells were seeded at 10The next step involved visualization of the cell nuclei .HEL299, HeLa, and OS 143B cells were seeded in a culture flask and cultured until gaining 70 % confluence. Next, the media were supplemented with cPt, TAMRA-TP10, and TAMRA-TP10 + cPt in concentrations of 10p < 0.05 was assumed to be statistically significant  was calculated. The data of fluorescent microscopy included minimum 3-time repeated experiments and those of contrast phase microscopy at least three times. Data were analyzed by Friedman ANOVA and Kendall Concordance using STATISTICA version 9.1 data analysis software system, and All tested cell lines were incubated with TAMRA-TP10 at concentrations in the range of 0.1–40 μM. The experiments were performed in order to select TAMRA-TP10 concentration for further research. The criterion of the selection was its lack of toxicity in each cell line .cPt at the concentration of 1 μM did not affect cell viability in non-cancer cell lines Fig. , b. ConsThe higher concentrations of the drug produced an evident inhibition of cell viability in the cancer cell lines Fig. , d. HighFigure All tested probes were compared to a sample without any compounds, used as a negative control.The action of TAMRA-TP10 + cPt on healthy cell viability Fig. , b was rThe response of the cancer cell lines to the treatment with TAMRA-TP10 + cPt. Figure To avoid the impact of TAMRA itself, the effect of the compounds in question (TP10 + cPt or TP10) without the dye was analyzed on one non-cancer Fig.  and one It turned out that their effects on both cell lines were comparable to those noticed after the complex with this dye or TP10 with it Fig. , f. WhatPTD4 + cPt (with TAMRA or without) as well as PTD4 (with TAMRA or without) did not indicate a statistically significant effect on the healthy cell viability Fig. . A lack TAMRA-TP10, TAMRA-TP10 + cPt, TAMRA-PTD4, and TAMRA-PTD4 + cPt.The ability of crossing the cell membrane by the compounds in question as well as their localization in the cell was verified by fluorescence microscopy. HEL299 and HeLa cells were incubated with Figure TAMRA-TP10 are presented in the next picture .TAMRA-TP10 + cPt, cPt, and TAMRA-TP10 were checked on one non-cancer cell line (HEL299) and two cancer  cell lines for 5 days. The results of this experiments are presented in the pictures bellow  may be a result of interactions which may occur between TP10 and cPt. These interactions may have a pharmacokinetic or pharmacodynamic dimension.The possible pharmacokinetic interactions may concern cellular bioavailability. TP10 could intensify the transport of cPt through the cellular membrane and thereby increase the internalization of this anticancer drug. Similar observations have been made by others with the usage of CPP conjugates with such small chemotherapeutic anticancer drugs as doxorubicin, methotrexate, or paclitaxel  could be additive to the one performed by cPt.The anticancer activity of TP10 does not seem to be very surprising as certain CPPs together with TP10, apart from the ability to translocate the cargo, are known for other activities, including the antitumor one  cell lines. An explanation for this experimental fact may be a different access of the tested compounds to healthy or cancer cells. Such a difference in cellular access was visualized by fluorescent microscopy, which showed a lack of internalization of TP10 and PTD4 in the former ones. Thus, it may be assumed that these compounds indicate a kind of selectivity to the cancer cell lines. A similar phenomenon was reported by Song et al. It is not known which factors provide the specificity of cationic peptides for the cancer cell membranes. However, certain features of these membranes have been pointed out as possible reasons, e.g., different membrane composition and fluidity, more negative charge, higher transmembrane potential, and increased level of acidic components on the surface  in the chemical structure of TP10. Its role seems to be complex. As has been indicated, a reactive amino group at Lys is a suitable handle to connect this CPP to such cargos as hydrophilic macromolecules  and its individual components on one non-cancer and two cancer cell lines. This long-term effect has been estimated on the basis of inverted phase contrast microscopic images. As compared with TP10 or cPt, the most potent action on the cancer cells has become visible if the complex was applied. Thus, the presented here microscopic images may support the results obtained in the MTT test as well as the conclusions drawn on their basis.In summary, this study has demonstrated an improved anticancer effect of TP10 + cPt complex in comparison with that produced by cPt. This effect could result from pharmacokinetic and/or pharmacodynamic interactions between the complex’s components. Additionally, TP10 + cPt was relatively safe for the non-cancer cell lines. Although, these conclusions have been drawn only on the basis of a qualitative analysis, they may serve as grounds of further more detailed research which would open new clinical prospects for this old anticancer drug.


N--2-phenylacetamide derivatives (3a-3j) were synthesized via an amidation reaction using EDC and HOBt in acetonitrile solvent at room temperature condition. Chemical structures were characterized by 1H NMR, IR and MS spectroscopic methods and related melting points were also determined. The anticancer activity was evaluated using MTT procedure in-vitro. All compounds were tested against SKNMC (Neuroblastoma), HT-29 (Colon cancer) and PC3 (Prostate cancer) cell lines. According to the toxicological data, none of the synthesized derivatives exerted superior activity than doxorubicin as reference drug. Derivatives with Ortho chlorine (compound 3d), meta methoxy (compound 3h) and meta fluorine (compound 3b) substituents on the phenyl ring exhibited the best cytotoxic activity against SKNMC (IC50 = 4.5 ± 0.035 µM), HT-29 (IC50 = 3.1 ± 0.030 µM) and PC3 (IC50 = 12.6 ± 0.302 µM) cell lines respectively. A new series of  Cancer is a major problem throughout the world and is the second leading cause of mortality in developed countries. Since, many of the current treatments have problems with adverse effects and drug-resistance; there is a strong and vital demand for the discovery and development of effective new anticancer therapeutics. Currently, cancer is the second leading cause of death in the developed countries. Tremendous progress has been made in the war against cancer with the development of many novel chemotherapy agents. The main treatments for neoplastic diseases involve surgery, chemotherapy and radiotherapy. Chemotherapy involves the use of low-molecular-weight drugs  to selectively destroy tumor cells or at least limit their proliferation. Disadvantages of many anticancer agents include myelosuppression, gastrointestinal side effects (nausea and vomiting), hair loss , and also the development of clinical resistance -7.During recent years a wide and intense investigation of different pharmacophores and chemical classes containing 1,3,4-thiadiazole have been carried out. Many of these derivatives possess interesting and potential biological effects such as antimicrobial, antitubercular, antiviral, anti-inflammatory, anticonvulsant, antihypertensive, antioxidant, antifungal and anticancer activity and now there are in the market as common used drugs  8-16).-16.8-16)etc , HT-29 (Colon cancer) and SKNMC (Neuroblastoma). ChemistryAccording to the MTT assaymeta (compound 3b) demonstrated the best anticancer activity against SKNMC cell line. This pattern was also observed for PC3 cell line. But, para substitution (compound 3c) of fluorine afforded a better activity against HT-29 cell line. Chlorine moiety at position 2 of the phenyl ring rendered a high anticancer potency against SKNMC cell line (IC50 = 4.5 ± 0.035 µM). Substitution of the methoxy group on the phenyl ring enhanced the activity against HT-29 cell line especially at positions ortho and meta (compound 3g and 3h). Ortho position of the methoxy moiety was also favorable for cytotoxic activity against SKNMC and PC3 cell lines (compound 3 g). Substitution of bromine atom at position 4 of the phenyl ring caused a better anticancer activity against SKNMC cell line compared to HT-29 and PC3 cell line. According to the Chemistry254 plates were applied for analytical TLC. Column chromatography was performed on Merck silica gel (70-230 mesh) for purification of intermediate and final compounds. 1H-NMR spectra were recorded using a Varian 400 MHz and Bruker 200 MHz spectrometer, and chemical shifts are expressed as δ (ppm) with tetramethylsilane (TMS) as internal standard. The IR spectra were obtained on a Shimadzu 470 spectrophotometer (potassium bromide disks). Melting points were determined using electrothermal melting point analyzer apparatus and are uncorrected. The mass spectra were run on a Finigan TSQ-70 spectrometer  at 70 eV. All cell lines were purchased from Pasteur Institute of Iran.All starting materials, reagents and solvents were purchased from commercial suppliers like Merck and Aldrich companies. The purity of the prepared compounds was proved by thin layer chromatography (TLC) using various solvents of different polarities. Merck silica gel 60 FGeneral procedure for synthesis of N--2-phenylacetamide derivatives (3a-3j)N-ethyl-N-dimethylaminopropyl carbodiimide (EDC) and hydroxybenzotriazole (HOBt) in acetonitrile solvent, and was stirred for 30 minutes at room temperature. Then, Equimolar quantity of 5-amino-1,3,4-thiadiazole-2-thiol (n-hexane and purified by column chromatography (EtOAc/Petroleum ether).According to the 2-(2-Fluorophenyl)-N-acetamide (3a)0C, Yield: 63%, IR  ῡ: 3151, 2931, 2804, 1712, 1570, 1492, 1465, 1342, 1311, 1234, 1180, 1145, 1068, 759, 694. MS : 296 , 133 (48), 109 (100), 83 (15). mp. 224 2-(3-Fluorophenyl)-N-acetamide (3b)0C, Yield: 78%, IR  ῡ: 3147, 2924, 1708, 1570, 1485, 1458, 1307, 1253, 1138, 1068, 964, 767. MS : 296 , 133 (55), 109 (100), 83 (10). mp. 162 2-(4-Fluorophenyl)-N-acetamide (3c)0C, Yield: 72%, 1H NMR  δ: 3.1 , 3.71 , 7.01 , 7.30 , 12.3 . IR  ῡ: 3140, 3100, 2924, 1963, 1554, 1504, 1296, 1220, 1120, 1062. MS : 269 , 133 (55), 109 (100), 83 (30), 59 (10). mp. 158-160 2-(2-Chlorophenyl)-N-acetamide (3d):0C, Yield: 82%, 1H NMR  δ: 3.86 , 3.98 , 7.26-7.58 , 7.85-7.91 , 10.15 . IR  ῡ: 3147, 2924, 2862, 1708, 1570, 1469, 1307, 1145, 1064, 744. mp. 212-216 2-(3-Chlorophenyl)-N-acetamide(3e)0C, Yield: 46%, 1H NMR  δ: 3.68 , 3.81 , 7.48-7.52 , 7.49 , 7.81 , 7.87 , 11.22 . IR  ῡ: 3151, 2927, 2804, 1701, 1573, 1469, 1311, 1222, 1153, 1064, 783, 682. mp. 210 2-(4-Chlorophenyl)-N-acetamide(3f)0C, Yield: 65%, 1H NMR  δ: 3.73 , 4.86 , 7.14-8.29 , 13.21 . IR  ῡ: 3444, 3137, 3095, 2850, 2718, 1687, 1551, 1519, 1492, 1352, 1089, 960, 818, 712. MS : 285 , 280 (45), 167 (94), 149 (100), 125 (12), 113 (15), 71 (20), 57 (20). mp. 174 N--2-(2-methoxyphenyl)acetamide(3g)0C, Yield: 47%, 1H NMR  δ: 3.82 , 3.89 , 3.95 , 7.02 , 7.27-7.4 , 11.44 .IR  ῡ: 3468, 3147, 2931, 2835, 1701, 1570, 1492, 1462, 1303, 1246, 1145, 1064, 1026, 798, 752. MS : 281 , 148 (75), 133 (15), 121 (100), 91 (90), 65 (15).mp. 200 N--2-(3-methoxyphenyl)acetamide(3h)0C, Yield: 65%, 1H NMR  δ: 3.67 , 3.79 , 3.84 , 6.88 , 7.26-7.33 , 7.29 , 11.24 . IR  ῡ: 3410, 3147, 2927, 1701, 1570, 1489, 1465, 1307, 1261, 1149, 1064, 779, 759, 690.mp. 114 N--2-(4-methoxyphenyl)acetamide(3i)0C, Yield: 79%, 1H NMR  δ: 3.35 , 3.38 , 3.79 , 7.76 , 8.02 , 13.1 . MS : 281 , 238 (25), 148 (90), 121 (100), 91 (25), 78 (30). mp. 152 2-(4-Bromophenyl)-N-acetamide(3j)0C, Yield: 68%, 1H NMR  δ: 3.67 , 3.75 , 7.18 , 7.47 , 10.6 . IR  ῡ: 3150, 2980, 2825, 1699, 1571, 1558, 1487, 1400, 1319, 1296, 1064, 1012, 788. MS : 331 , 329 , 198 (30), 196 (30), 171 (60), 169 (60), 133 (90), 89 (100), 63 (30).mp. 198 Cytotoxicity assayoC were done, and then the medium was discarded. Dimethyl sulfoxide (60 μL) was added to each well, and the solution was vigorously mixed to dissolve the purple tetrazolium crystals. The absorbance of each well was measured by plate reader  at a test wavelength of 550 nm against a standard reference solution at 690 nm. The amount of produced purple formazan is proportional to the number of viable cells  were tested for cytotoxic activity at 0.1-500 mcg/mL concentration in three human cancer cell lines of PC3 cell (prostate cancer), HT-29 (Colon cancer) and SKNMC (Neuroblastoma). Cells from different cell lines were seeded in 96-well plates at the density of 8000–10,000 viable cells per well and incubated for 48 hours to allow cell attachment. The cells were then incubated for another 48-96 hours (depends to cell cycle of each cell line) with various concentrations of compounds 3a-3j. Cells were then washed in PBS, and 20 μL of MTT -2,5-diphenyl tetrazolium bromide solution (5 mg/mL) were added to each well. An additional 4 hours of incubation at 37 le cells .


Apis mellifera from various tree buds) exhibits a broad spectrum of biological activities. Recently, in vitro and in vivo data suggest that propolis has anticancer properties, but is the cytoxicity of propolis specific for tumor cells? To answer this question, the cytotoxicity of phenolic extracts from Portuguese propolis of different origins was evaluated using human tumor cell lines , and non-tumor primary cells (PLP2). The studied propolis presented high cytotoxic potential for human tumor cell lines, mostly for HCT15. Nevertheless, excluding HCT15 cell line, the extracts at the GI50 obtained for tumor cell lines showed, in general, cytotoxicity for normal cells (PLP2). Propolis phenolic extracts comprise phytochemicals that should be further studied for their bioactive properties against human colon carcinoma. In the other cases, the proximity of the in vitro cytotoxic doses for tumor and normal cell lines should be confirmed by in vivo tests and may highlight the need for selection of specific compounds within the propolis extract.With a complex chemical composition rich in phenolic compounds, propolis (resinous substance collected by  Epidemiological data support the concept that natural products, in special, phenols and polyphenols in diet are safe and nontoxic and have long-lasting beneficial effects on human health. Among the many biological properties presented by them, the ones involved with cancer therapy are remarkable .Many plants rich in polyphenols have been shown to display interesting anticancer effects on cell lines and murine models, yielding higher effects when compared to pure natural or synthetic compounds . ProductPropolis is a chemically complex product obtained by bees from the resinous exudates of leaf buds, shoots, and petioles of leaves of different plants present around the hive, which is mixed with wax and salivary secretions. In the hive it has a multifunctional role as a material for construction, maintenance, and defense . Named a in vitro and in vivo assays it was possible to define different mechanisms of actions for the bee glue and its components, such as suppressing cancer cells proliferation via its anti-inflammatory effects, decreasing the cancer stem cell populations, blocking specific oncogene signaling pathways, exerting antiangiogenic effects, and modulating the tumor microenvironment [Propolis has a wide spectrum of biological and pharmacological properties, which have been intensively researched in cell and animal experiments and thus it can be considered as a drug from the beehive . Recentlironment –11.n), providing identification of seventy-six phenolic compounds [ in vitro antitumor activity of a propolis sample from Azores archipelago was evaluated on colon carcinoma cell line HCT15 presenting cytotoxicity in a dose- and time-dependent way [Propolis phenolic profile from different Portuguese regions was recently characterized and quantified through high-pressure liquid chromatography (HPLC) and liquid chromatography with diode-array detection coupled to electrospray ionization tandem mass spectrometry  and nontumor primary cells (PLP2).The study was performed on propolis samples collected from five different geographical regions in Portugal: north , central coast , south , Azores Archipelago , and Madeira Island . All the samples were obtained between 2007 and 2009 after the honey harvesting season (July/September), by conventional scraping or through plastic screens. These propolis samples were then stored at −20°C until analysis.50 (extract concentration that inhibited 50% of the net cell growth) values. Ellipticine was used as positive control.The extraction was made according to the work previously described . Prior tFetal bovine serum (FBS), L-glutamine, Hank's balanced salt solution (HBSS), trypsin-EDTA (ethylenediaminetetraacetic acid), penicillin/streptomycin solution , RPMI-1640, and DMEM media were from Hyclone . Acetic acid, ellipticine, sulforhodamine B (SRB), trypan blue, trichloroacetic acid (TCA), and Tris were from Sigma Chemical Co. . Water was treated in a Milli-Q water purification system .2. Each cell line was plated at an appropriate density  in 96-well plates for 24 h. Cells were then treated for 48 h with various extract concentrations. Following this incubation period, the adherent cells were fixed by adding cold 10% trichloroacetic acid  and incubated for 60 min at 4°C. Plates were then washed with deionized water and dried; sulforhodamine B solution  was then added to each plate well and incubated for 30 min at room temperature. Unbound SRB was removed by washing with 1% acetic acid. Plates were air-dried, the bound SRB was solubilised with 10 mM Tris (200 μL) and the absorbance was measured at 540 nm in ELX800 Microplate Reader  [Five human tumor cell lines were used: MCF-7 (breast adenocarcinoma) from DSMZ (Leibniz-Institut DSMZ—Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH), NCI-H460 , HCT15 (colon carcinoma), HeLa , and HepG2 (hepatocellular carcinoma) from European collection of cell cultures (ECACC). Cells were routinely maintained as adherent cell cultures in RPMI-1640 medium containing 10% heat-inactivated FBS  and 2 mM glutamine or in DMEM supplemented with 10% FBS, 2 mM glutamine, 100 U/mL penicillin, and 100 mg/mL streptomycin (HeLa and HepG2 cells), at 37°C, in a humidified air incubator containing 5% COki, USA) .μg/mL streptomycin and divided into 1 × 1 mm3 explants. Some of these explants were placed in 25 cm2 tissue flasks in DMEM medium supplemented with 10% fetal bovine serum, 2 mM nonessential amino acids, 100 U/mL penicillin, and 100 mg/mL streptomycin and incubated at 37°C with a humidified atmosphere containing 5% CO2. The medium was changed every two days. Cultivation of the cells was continued with direct monitoring every two to three days using a phase contrast microscope. Before confluence was reached, cells were subcultured and plated in 96-well plates at a density of 1.0 × 104 cells/well and cultivated in DMEM medium with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin [A cell culture was prepared from a freshly harvested porcine liver obtained from a local slaughter house, and it was designed as PLP2. Briefly, the liver tissues were rinsed in Hank's balanced salt solution containing 100 U/mL penicillin and 100 ptomycin .n = 3) were analysed and all the assays were carried out in triplicate. The results are expressed as mean values and standard deviation (SD). The differences between the different samples were analysed using one-way analysis of variance (ANOVA) followed by Tukey's honestly significant difference post hoc test with α = 0.05, coupled with Welch's statistic. This treatment was carried out using SPSS v. 18.0 program. The regression analysis between phenolic acids, phenolic esters, total phenolics, flavonols, flavones, dihydroflavonols, flavanones, flavonoid esters, total flavonoids (mg/g of extract), and cytotoxicity GI50 values (μg/mL) was performed using the same statistical package.For all the experiments three samples  and in a porcine liver primary cell culture (PLP2), established by us. All the studied extracts inhibited the growth of the mentioned tumor cell lines. N1 gave the highest cytotoxicity, followed by A2 , presents et al.  This act50 values for MCF7 cell line were not very different from the extracts N1 or A2, which can be due to the similar high content in total flavonoids and the specificity of this cell line to flavonoids. This sample presented the major concentration of flavones and flavanones, two important groups of compounds regarding biological activity.Despite the lower concentration in total phenolics presented in the sample N8, the GI50 values for all the tested tumor cell lines. This fact could be explained by its poor chemical composition  and, consequently, with total phenolic (R2 higher than 0.5) contents but not with phenolic esters (R2 lower than 0.5). NCI-H460 cell line presented the highest correlation for this class of compounds (R2 = 0.9454).The correlations established between the chemical composition and the cytotoxicity results are presented in R2 values higher than 0.5) with the cytotoxicity observed against MCF7, NCI-H460, HCT15, and HeLa cell lines. However, the cytotoxicity was not correlated  with flavonols, dihydroflavonols, and flavonoid esters. The cytotoxicity observed in HCT15 cell line was the one that correlates better with the composition in phenolic compounds.Total flavonoids were positively correlated  liver primary culture (PLP2), with GI50 values close to those obtained for tumor cell lines. The cytotoxicity for PLP2 primary culture was only positively correlated with phenolic acids (R2 = 0.7442) and consequently with total phenolics (R2 = 0.6207).The evaluation of the cytotoxicity using PLP2 is very important since mammalian hepatocytes still represent an obligatory step in the evaluation of toxic compounds that lead to the production of various metabolites, which are the ultimate cause of toxicity. We used porcine liver as anThe colon carcinoma cells, HCT15, seem to be the most promising to be controlled by the studied propolis extracts since, with exception of sample S2, all the cytotoxicity levels for the tumor cells are significantly above the ones found for nontumor PLP2 cells. The same behaviour was observed for the NCI-H460 cell line; however, in this case the cytotoxicity levels for tumor and nontumor cells are close which reduces the selectivity. The sample from Madeira showed the lowest cytotoxicity for all the tested tumor cell lines, which indicates that the chemical composition of the samples has a significant influence on their bioactivity, since this sample presented a quite specific phenolic profile, only with flavones.The highest cytotoxicity revealed by samples N1 and A2 against all the tested tumor cell lines is certainly related to their higher concentrations in total phenolics and phenolic acids. Phenolic acids and analogues are, in fact, known to display a wide variety of biological functions, in addition to their primary antioxidant activity, which are mainly related to modulation of carcinogenesis , 19. IndThe similar toxicity for MCF7 cell line observed between N8, N1, and A2 can be explained by the similar high content in total flavonoids, presenting N8 as the highest concentration of flavones and flavanones. The most common compounds within the flavones were apigenin, chrysin, and luteolin. They are responsible for main benefits to human health including anticarcinogenic effects . In the The present data highlights the high cytotoxicity of Portuguese propolis against tumor cell lines, being in agreement with Búfalo et al. , who rep50 value for Hek-293 when compared to tumor cell lines.Despite the high cytotoxicity displayed by most of the propolis samples against tumor cell lines studied herein, the samples also showed toxicity for nontumour  liver primary culture (PLP2). da Silva Frozza et al.  have als in vitro cytotoxic doses for tumor and normal cell lines should be taken into consideration and confirmed by in vivo tests. Additional research is needed to determine which compounds are involved in cytotoxicity against normal cells in order to develop new propolis formulations without those compounds to be selectively used against tumor cells.Propolis phenolic extracts comprise phytochemicals that should be further studied for their bioactive properties against human colon carcinoma. In the other cases, the proximity of the