Bispecific Antibodies for IFN-β Delivery to ErbB2+ Tumors

The main aim of our work was to create a full-length bispecific antibody (BsAb) as a vehicle for the targeted delivery of interferon-beta (IFN-β) to ErbB2+ tumor cells in the form of non-covalent complex of BsAb and IFN-β. Such a construct is a CrossMab-type BsAb, consisting of an ErbB2-recognizing trastuzumab moiety, a part of chimeric antibody to IFN-β, and human IgG1 Fc domain carrying knob-into-hole amino acid substitutions necessary for the proper assembly of bispecific molecules. The IFN-β- recognizing arm of BsAb not only forms a complex with the cytokine but neutralizes its activity, thus providing a mechanism to avoid the side effects of the systemic action of IFN-β by blocking IFN-β Interaction with cell receptors in the process of cytokine delivery to tumor sites. Enzyme sandwich immunoassay confirmed the ability of BsAb to bind to human IFN-β comparable to that of the parental chimeric mAb. The BsAb binds to the recombinant ErbB2 receptor, as well as to lysates of ErbB2+ tumor cell lines. The inhibition of the antiproliferative effect of IFN-β by BsAb (IC50 = 49,3 µg/mL) was demonstrated on the HT29 cell line. It can be proposed that the BsAb obtained can serve as a component of the immunocytokine complex for the delivery of IFN-β to ErbB2-associated tumor cells.

Study of the effect of Pioglitazone and Cetuximab on Cancer Stem-like Cellsenriched HT29 cell line

One of the major causes of cancer resistance to chemotherapy has been found to be the presence of Cancer Stem Cells (CSCs) in cancerous tissues. Probably, these cells are the source of cancer and the cause of malignancy and recurrence in the affected population. Therefore, it is possible to target CSCs to treat cancer. Since the percentage of CSCs in the total tumor mass is very low, so studies about these cells depend on their isolation and enrichment methods. Some studies have suggested that EMT induction in population of normal epithelial cells and cancer cells by inhibiting of E-cadherin, protects them against chemotherapy, anticancer and apoptosis drugs, moreover they get characteristics of CSCs. So in order to study CSCs can enrich them by inhibiting of E-cadherin in tumor population.In this study, we tried to examine how the effect of Pioglitazone and Cetuximab, two drugs used in chemotherapy of Colon Cancer, on CSCs enriched HT29 cell line (was called HT29-shE) in which CSCs were enriched with induction of EMT by inhibiting of E-cadherin using shRNA.For this purpose, after cell preparation EMT and CSCs markers in Pioglitazone and Cetuximab treated cells were assessed and compared with untreated cells using flow cytometry, real‐time PCR and microscopic monitoring. The findings showed mesenchymal morphology of HT29-shE changed to epithelial morphology after Pioglitazone and Cetuximab treatment, moreover E-cadherin expression increased and Vimentin expression decreased. In addition, expression of CSC markers (CD133+ and CD44+) were reduced in HT29-shE after treatment.

Effect of the Cellular Extract of Co-cultured Lactobacillus Casei on BAX and Human β-Defensin 2 Genes Expression in HT29 Cells

Aims: Defensins are cysteine-rich antimicrobial cationic peptides and BAX is a proapoptotic gene that can cause cell death. This study aimed to investigate the effect of cellular extract of co-cultured Lactobacillus casei on the expression of BAX and human β-defensin 2 (hBD-2) genes in HT29 cells. Methods & Materials: This experimental study was conducted in the Research Center for Pharmaceutical Nanotechnology of Tabriz University of Medical Sciences in 2017. The HT29 cell line was obtained from the Pasteur Institute of Iran, and cells were assessed using Microculture Tetrazolium Test (MTT) after culturing. DNA was extracted from the treated cells, and then the DNA ladder assay was carried out. After preparing cDNA, the expression levels of BAX and hBD-2 genes in the HT29 cell line were measured using a real-time Polymerase Chain Reaction (PCR) method. Findings: The results of the MTT assay indicated that Lactobacillus casei inhibited the proliferation of HT29 cells and induced apoptosis in these cells. Results of DAPI staining and DNA ladder assay obtained from treating HT29 cells by Lactobacillus casei showed qualitative changes in cell apoptosis. Moreover, realtime PCR results indicated that Lactobacillus casei bacteria significantly increased the expression of the hBD-2 gene in HT29 colon cancer cells within 12-24 hours (P= 0.023), while BAX gene expression showed no significant change in the first 24 hours (P= 0.37). Conclusion: The extract of Lactobacillus casei can be used to stimulate cancer cells to produce β-defensins, inhibit pathogens, prevent the stimulation of cellular signaling, and fight antibiotic-resistant bacteria.

Unveil the Anticancer Potential of Limomene Based Therapeutic Deep Eutectic Solvents

Deep eutectic solvents have been recently reported as an interesting alternative to improve the therapeutic efficacy of conventional drugs, hence called therapeutic deep eutectic solvents (THEDES). The main objective of this work was to evaluate the potential of limonene (LIM) based THEDES as new possible systems for cancer treatment. LIM is known to have antitumor activity, however it is highly toxic and cell viability is often compromised, thus this compound is not selective towards cancer cells. Different THEDES based on LIM were developed to unravel the anticancer potential of such systems. THEDES were prepared by gently mixing saturated fatty acids menthol or ibuprofen (IBU) with LIM. Successful THEDES were obtained for Menthol:LIM (1:1), CA:LIM (1:1), IBU:LIM (1:4) and IBU:LIM(1:8). The results indicate that all the THEDES present antiproliferative properties, but IBU:LIM (1:4) was the only formulation able to inhibit HT29 proliferation without comprising cell viability. Therefore, IBU:LIM (1:4) was the formulation selected for further assessment of anticancer properties. The results suggest that the mechanism of action of LIM:IBU (1:4) is different from isolated IBU and LIM, which suggest the synergetic effect of DES. In this work, we unravel a methodology to tune the selectivity of LIM towards HT29 cell line without compromising cell viability of healthy cells. We demonstrate furthermore that coupling LIM with IBU leads also to an enhancement of the anti-inflammatory activity of IBU, which may be important in anti-cancer therapies.

Synthesis of 9-Hydroxystearic Acid Derivatives and Their Antiproliferative Activity on HT 29 Cancer Cells

9-Hydroxystearic acid (9-HSA) is an endogenous cellular lipid that possesses antiproliferative and selective effects against cancer cells. A series of derivatives were synthesized in order to investigate the effect of the substituent in position 9 and on the methyl ester functionality on the biological activity. The two separate enantiomers of methyl 9-hydroxystearate and of methyl 9-aminostearate showed antiproliferative activity against the HT29 cell line. This indicates the importance of position 9 groups being able to make hydrogen bonding with the molecular target. Further, this effect must be preserved when the carboxy group of 9-HSA is esterified. The biological tests showed that the amines, contrarily to methyl esters, resulted in cytotoxicity. A deep investigation on the effect of methyl (R)-9-hydroxystearate on HT29 cells showed an antiproliferative effect acting through the CDKN1A and MYCBP gene expression.