Expression of GA733-Fc Fusion Protein as a Vaccine Candidate for Colorectal Cancer in Transgenic Plants

The tumor-associated antigen GA733 is a cell-surface glycoprotein highly expressed in colorectal carcinomas. In this study, 3 recombinant genes were constructed as follows: GA733 tagged to the ER retention sequence KDEL (GA733K), GA733 fused to the immunoglobulin Fc fragment (GA733-Fc), and GA733-Fc fused to the ER retention sequence (GA733-FcK). Agrobacterium-mediated transformation was used to generate transgenic plants expressing recombinant genes. The presence of transgenes was confirmed by genomic PCR. Western blot, confocal immunofluorescence, and sandwich ELISA showed the expression of recombinant proteins. The stability, flexibility, and bioactivity of recombinant proteins were analyzed and demonstrated through N-glycosylation analysis, animal trials, and sera ELISA. Our results suggest that the KDEL retained proteins in ER with oligomannose glycan structure and enhanced protein accumulation level. The sera of mice immunized with GA733-FcK purified from plants contained immunoglobulins which were at least as efficient as the mammalian-derived GA733-Fc at recognizing human colorectal cancer cell lines. Thus, a plant system can be used to express the KDEL fusion protein with oligomannose glycosylation, and this protein induces an immune response which is comparable to non-KDEL-tagged, mammalian-derived proteins

Effects of endocrine disrupting chemicals on expression of phospholipid hydroperoxide glutathione peroxidase mRNA in rat testes

Phospholipid hydroperoxide glutathione peroxidase (PHGPx), an antioxidative selenoprotein, is modulated by estrogen in the testis and oviduct. To examine whether potential endocrine disrupting chemicals (EDCs) affect the microenvironment of the testes, the expression patterns of PHGPx mRNA and histological changes were analyzed in 5-week-old Sprague-Dawley male rats exposed to several EDCs such as an androgenic compound [testosterone (50, 200, and 1,000 µg/kg)], anti-androgenic compounds [flutamide (1, 5, and 25 mg/kg), ketoconazole (0.2 and 1 mg/kg), and diethylhexyl phthalate (10, 50, and 250 mg/kg)], and estrogenic compounds [nonylphenol (10, 50, 100, and 250 mg/kg), octylphenol (10, 50, and 250 mg/kg), and diethylstilbestrol (10, 20, and 40 µg/kg)] daily for 3 weeks via oral administration. Mild proliferation of germ cells and hyperplasia of interstitial cells were observed in the testes of the flutamide-treated group and deletion of the germinal epithelium and sloughing of germ cells were observed in testes of the diethylstilbestrol-treated group. Treatment with testosterone was shown to slightly decrease PHGPx mRNA levels in testes by the reverse transcriptionpolymerase chain reaction. However, anti-androgenic compounds (flutamide, ketoconazole, and diethylhexyl phthalate) and estrogenic compounds (nonylphenol, octylphenol, and diethylstilbestrol) significantly upregulated PHGPx mRNA in the testes (p < 0.05). These findings indicate that the EDCs might have a detrimental effect on spermatogenesis via abnormal enhancement of PHGPx expression in testes and that PHGPx is useful as a biomarker for toxicity screening of estrogenic or antiandrogenic EDCs in testes

Ganglioside GM1 influences the proliferation rate of mouse induced pluripotent stem cells

Gangliosides play important roles in the control of severalbiological processes, including proliferation and transmembranesignaling. In this study, we demonstrate the effect ofganglioside GM1 on the proliferation of mouse inducedpluripotent stem cells (miPSCs). The proliferation rate ofmiPSCs was lower than in mouse embryonic stem cells(mESCs). Fluorescence activated cell sorting analysis showedthat the percentage of cells in the G2/M phase in miPSCs waslower than that in mESCs. GM1 was expressed in mESCs, butnot miPSCs. To confirm the role of GM1 in miPSC proliferation,miPSCs were treated with GM1. GM1-treated miPSCsexhibited increased cell proliferation and a larger number ofcells in the G2/M phase. Furthermore, phosphorylation ofmitogen-activated protein kinases was increased in GM1-treated miPSCs

Production of Monoclonal Antibodies in Plants for Cancer Immunotherapy

Plants are considered as an alternative platform for recombinant monoclonal antibody (mAb) production due to the improvement and diversification of transgenic techniques. The diversity of plant species offers a multitude of possibilities for the valorization of genetic resources. Moreover, plants can be propagated indefinitely, providing cheap biomass production on a large scale in controlled conditions. Thus, recent studies have shown the successful development of plant systems for the production of mAbs for cancer immunotherapy. However, their several limitations have to be resolved for efficient antibody production in plants

7,8-Dihydroxyflavone induces mitochondrial apoptosis and down-regulates the expression of ganglioside GD3 in malignant melanoma cells

Abstract Malignant melanoma is a skin cancer with poor prognosis and high resistance to conventional treatment. 7,8-dihydroxyflavone (7,8-DHF) has shown anti-carcinogenic, anti-inflammatory, anti-oxidant, and pharmacological effects in several types of cancer. However, the relationship between ganglioside expression and the anti-cancer effects of 7,8-DHF in melanoma is not fully understood. In the present study, 7,8-DHF exhibits specific anti-proliferation, anti-migration, and G2/M phase cell-cycle arrest effects on both melanoma cancer cell lines, and induces mitochondrial dysfunction and apoptosis, making it a potent candidate for anti-melanoma treatment. Furthermore, we confirmed that 7,8-DHF significantly reduces the expression levels of ganglioside GD3 and its synthase, which are known to be closely involved in carcinogenesis. Taken together, our findings suggest that 7,8-DHF may be a potent anti-cancer drug candidate for the treatment of malignant melanoma

GM1 Induced the inflammatory response related to the Raf-1/MEK1/2/ERK1/2 pathway in co-culture of pig mesenchymal stem cells with RAW264.7

Pig-human xenotransplantation can trigger cell-mediated immune responses. We explored the role of gangliosides in inflammation related to immune rejection in xenotransplantation. Co-culture of xenogeneic cells (pig-MSCs and RAW264.7) was used to emulate xenotransplantation conditions. MTT assay results indicated that cell viability was significantly decreased in pADMSCs co-cultured with RAW264.7 cells. GM1 and GM3 were highly expressed in pADMSCs co-cultured with RAW264.7 cells. pADMSCs co-cultured with RAW264.7 cells strongly expressed pro-inflammatory proteins such as COX-2, iNOS, p50, p65, pIκBα, and TNF-α. GM1-knockdown pADMSCs co-cultured with RAW 264.7 cells did not show significantly altered cell viability, but pro-inflammatory proteins were markedly inhibited. Co-culture of pADMSCs with RAW264.7 cells induced significant phosphorylation (p) of JNK1/2 and pERK1/2. However, pERK1/2 and pJNK1/2 were decreased and MEK1/2 and Raf1 were suppressed in GM1-knockdown pADMSCs co-cultured with RAW264.7 cells. Thus, the Raf-1/MEK1/2/ERK1/2 and JNK1/2 pathways were significantly upregulated in response to increases of GM1 in co-cultured xenogeneic cells. However, the inflammatory response was suppressed in co-culture of GM1-knockdown pADMSCs with RAW264.7 cells via down-regulation of the Raf-1/MEK1/2/ERK1/2 and JNK1/2 pathways. Therefore, the ganglioside GM1 appears to play a major role in the inflammatory response in xenotransplantation via the Raf-1/MEK1/2/ERK1/2 and JNK1/2 pathways

Application of Mesenchymal Stem Cells in Inflammatory and Fibrotic Diseases

Mesenchymal stem cells (MSCs) are multipotent stem cells that can be isolated from various tissues in the adult body. MSCs should be characterized by three criteria for regenerative medicine. MSCs must (1) adhere to plastic surfaces, (2) express specific surface antigens, and (3) differentiate into mesodermal lineages, including chondrocytes, osteoblasts, and adipocytes, in vitro. Interestingly, MSCs have immunomodulatory features and secrete trophic factors and immune receptors that regulate the microenvironment in host tissue. These specific and unique therapeutic properties make MSCs ideal as therapeutic agents in vivo. Specifically, pre-clinical and clinical investigators generated inflammatory and fibrotic diseases models, and then transplantation of MSCs into diseases models for therapeutic effects investigation. In this review, we characterize MSCs from various tissues and describe their applications for treating various inflammation and fibrotic diseases