Previously, we have succeeded in converting induced pluripotent stem cells (iPSCs) into cancer stem cells (CSCs) by treating the iPSCs with conditioned medium of Lewis lung carcinoma (LLC) cells. The converted CSCs, named miPS-LLCcm cells, exhibited the self-renewal, differentiation potential, and potential to form malignant tumors with metastasis. In this study, we further characterized miPS-LLCcm cells both in vivo and in vitro. The tumors formed by subcutaneous injection showed the structures with pathophysiological features consisting of undifferentiated and malignant phenotypes generally found in adenocarcinoma. Metastasis in the lung was also observed as nodule structures. Excising from the tumors, primary cultured cells from the tumor and the nodule showed self-renewal, differentiation potential as well as tumor forming ability, which are the essential characters of CSCs. We then characterized the epigenetic regulation occurring in the CSCs. By comparing the DNA methylation level of CG rich regions, the differentially methylated regions (DMRs) were evaluated in all stages of CSCs when compared with the parental iPSCs. In DMRs, hypomethylation was found superior to hypermethylation in the miPS-LLCcm cells and its derivatives. The hypo- and hypermethylated genes were used to nominate KEGG pathways related with CSC. As a result, several categories were defined in the KEGG pathways from which most related with cancers, significant and high expression of components was PI3K-AKT signaling pathway. Simultaneously, the AKT activation was also confirmed in the CSCs. The PI3K-Akt signaling pathway should be an important pathway for the CSCs established by the treatment with conditioned medium of LLC cells.

The Tridax procumbens flavonoids (TPF), are well known for their medicinal properties among local natives. The TPF are traditionally used for dropsy, anaemia, arthritis, gout, asthma, ulcer, piles, and urinary problems. It also used in treating gastric problems, body pain, and rheumatic pains of joints. The TPF have been reported to increase osteogenic functioning in mesenchymal stem cells. However, their effects on osteoclastogenesis remain unclear. The TPF isolated from T. procumbens and investigated the effects of the TPF inhibit on osteoclast differentiation and bone resorption activities using primary osteoclastic cells. Osteoclast formation was assessed by counting the number of tartrate resistant acid phosphatase (TRAP) positive multinucleated cells and by measuring both TRAP activities. The TPF significantly suppressed the RANKL-induced differentiation of osteoclasts and the formation of pits in primary osteoclastic cells. The TPF also decreased the expression of mRNAs related to osteoclast differentiation, including Trap, Cathepsin K, Mmp-9, and Mmp-13 in primary osteoclastic cells. The treatment of primary osteoclastic cells with the TPF decreased Cathepsin K, Mmp-9, and Mmp-13 proteins expression in primary osteoclastic cells. These results indicated that TPF inhibit osteoclastogenesis and pits formation activities. Our results suggest that the TPF could be a potential anti-bone resorptic agent to treat patients with bone loss-associated diseases such as osteoporosis.

“Combination therapy”, which is a treatment modality combining two or more therapeutic agents, is considered a cornerstone of cancer therapy. The combination of anticancer drugs, of which functions are different from the other, enhances the efficiency compared to the monotherapy because it targets cancer cells in a synergistic or an additive manner. In this study, the combination of paclitaxel and sorafenib in low concentration was evaluated to target cancer stem cells, miPS-BT549cmP and miPS-Huh7cmP cells, developed from mouse induced pluripotent stem cells. The synergistic effect of paclitaxel and sorafenib on cancer stem cells was assessed by the inhibition of proliferation, self-renewal, colony formation, and differentiation. While the IC₅₀ values of paclitaxel and sorafenib were approximately ranging between 250 and 300 nM and between 6.5 and 8 µM, respectively, IC₅₀ of paclitaxel reduced to 20 and 25 nM, which was not toxic in a single dose, in the presence of 1 µM sorafenib, which was not toxic to the cells. Then, the synergistic effect was further assessed for the potential of self-renewal of cancer stem cells by sphere formation ability. As a result, 1 µM of sorafenib significantly enhanced the effect of paclitaxel to suppress the number of spheres. Simultaneously, paclitaxel ranging in 1 to 4 nM significantly suppressed not only the colony formation but also the tube formation of the cancer stem cells in the presence of 1 µM sorafenib. These results suggest the combination therapy of paclitaxel and sorafenib in low doses should be an attractive approach to target cancer stem cells with fewer side effects.

Cripto-1 is a glycophosphatidylinositol (GPI) anchored signaling protein of epidermal growth factor (EGF)-Cripto-1-FRL1-Cryptic (CFC) family and plays a significant role in the early developmental stages and in the different types of cancer cells, epithelial to mesenchymal transition and tumor angiogenesis. Previously, we have developed cancer stem cells (miPS-LLCcm) from mouse iPSCs by culturing them in the presence of conditioned medium of Lewis Lung Carcinoma (LLC) cells for four weeks. Nodal and Cripto-1 were confirmed to be expressed in miPS-LLCcm cells by quantitative reverse transcription PCR (rt-qPCR) implying that Cr-1 was required in maintaining stemness. To investigate the biological effect of adding exogenous soluble CR-1 to the cancer stem cells, we have prepared a C-terminally truncated soluble form of recombinant human CR-1 protein (rhsfCR-1), in which the GPI anchored moiety was removed by substitution of a stop codon through site-directed mutagenesis. rhsfCR-1 effectively suppressed the proliferation and sphere forming ability of miPS-LLCcm cells in a dose-dependent manner in the range of 0 to 5 µg/mL, due to the suppression of Nodal-Cripto-1/ALK4/Smad2 signaling pathway. Frequency of sphere-forming cells was dropped from 1/40 to 1/69 by rhsfCR-1 at 1 µg/mL. Moreover, rhsfCR-1 in the range of 0 to 1 µg/mL also limited the differentiation of miPS-LLCcm cells into vascular endothelial cells probably due to the suppression of self-renewal, which should reduce the number of cells with stemness property. As demonstrated by a soluble form of exogenous Cripto-1 in this study, the efficient blockade would be an attractive way to study Cripto-1 dependent cancer stem cell properties for therapeutic application.

Background: Diabetes mellitus is the most occurring non-communicable disease resulting in a high blood glucose level. There has been an immense interest in the development of alternative medicines for diabetes treatment, specifically screening functional foods for phytochemicals with the capability of delaying or preventing glucose absorption through digestive enzymes (e.g. α-amylase) inhibition. So, the development of α-amylase inhibitors derived from natural food products is an alternative way to prevent diabetes mellitus. Objective: In this study, organic solvent extracts of the Arachis hypogaea (Peanut) and Cinnamomum tamala (Indian bay leaf/Tejpata) were used to investigate their potential α-amylase inhibition and cytotoxic activities through α-amylase inhibition assay and brine shrimp lethality bioassay respectively. Methods: The α-amylase inhibition assay was performed using the 3,5-dinitrosalicylic acid method for different concentrations of plant extracts. The optical density (OD) of the solutions were measured to determine the inhibition activity at 540 nm using a spectrophotometer. The cytotoxicity of the plant extracts was measured using brine shrimp (Artemia salina) lethality bioassay. Results: Among the different organic solvent extracts, peanut seed ethanol extract showed the highest α-amylase inhibition activity (67.68±8.67%) at 1.25 μg/mL concentration with an IC50 value of 0.61 μg/mL which is very close to standard α-amylase inhibitor Acarbose (72.34±4.23%) with an IC50 value of 0.32 μg/mL while acetone extract of Indian bay leaf exhibited the lowest inhibition activity (47.75±1.63%) with an IC50 value of 1.42 μg/mL at the same concentration. Besides, the maximum cytotoxic activity was found in acetone extract of peanut shell with an LC50 value of 57.87 μg/mL, whereas ethanol extract of peanut seed showed the lowest cytotoxicity with an LC50 value of 413.90 μg/mL. Conclusion: The result of the present work clearly indicates the potentiality of peanut seed ethanol extract to be used in the management of hyperglycemia as it significantly inhibits α-amylase activity while showing less cytotoxic activities.