GSK-3β regulates the endothelial-to-mesenchymal transition via reciprocal crosstalk between NSCLC cells and HUVECs in multicellular tumor spheroid models

Abstract Background Chemotherapy used for patients with unresectable lung tumors remains largely palliative due to chemoresistance, which may be due to tumor heterogeneity. Recently, multiple studies on the crosstalk between lung cancer cells and their tumor microenvironment (TME) have been conducted to understand and overcome chemoresistance in lung cancer. Methods In this study, we investigated the effect of reciprocal crosstalk between lung cancer cells and vascular endothelial cells using multicellular tumor spheroids (MCTSs) containing lung cancer cells and HUVECs. Results Secretomes from lung cancer spheroids significantly triggered the endothelial-to-mesenchymal transition (EndMT) process in HUVECs, compared to secretomes from monolayer-cultured lung cancer cells. Interestingly, expression of GSK-3β-targeted genes was altered in MCTSs and inhibition of this activity by a GSK-3β inhibitor induced reversion of EndMT in lung tumor microenvironments. Furthermore, we observed that HUVECs in MCTSs significantly increased the compactness of the spheroids and exhibited strong resistance against Gefitinib and Cisplatin, relative to fibroblasts, by facilitating the EndMT process in HUVECs. Subsequently, EndMT reversion contributed to control of chemoresistance, regardless of the levels of soluble transforming growth factor (TGF)-β. Using the MCTS xenograft mouse model, we demonstrated that inhibition of GSK-3β reduces lung cancer volume, and in combination with Gefitinib, has a synergistic effect on lung cancer therapy. Conclusion In summary, these findings suggest that targeting EndMT through GSK-3β inhibition in HUVECs might represent a promising therapeutic strategy for lung cancer therapy

HO-1089 and HO-1197, Novel Herbal Formulas, Have Antitumor Effects via Suppression of PLK1 (Polo-like Kinase 1) Expression in Hepatocellular Carcinoma

The treatment for hepatocellular carcinoma (HCC), a severe cancer with a very high mortality rate, begins with the surgical resection of the primary tumor. For metastasis or for tumors that cannot be resected, sorafenib, a multi-tyrosine protein kinase inhibitor, is usually the drug of choice. However, typically, neither resection nor sorafenib provides a cure. The drug discovery strategy for HCC therapy is shifting from monotherapies to combination regimens that combine an immuno-oncology agent with an angiogenesis inhibitor. Herbal formulas can be included in the combinations used for this personalized medicine approach. In this study, we evaluated the HCC anticancer efficacy of the new herbal formula, HO-1089. Treatment with HO-1089 inhibited HCC tumor growth by inducing DNA damage-mediated apoptosis and by arresting HCC cell replication during the G2/M phase. HO-1089 also attenuated the migratory capacity of HCC cells via the inhibition of the expression of EMT-related proteins. Biological pathways involved in metabolism and the mitotic cell cycle were suppressed in HO-1089-treated HCC cells. HO-1089 attenuated expression of the G2/M phase regulatory protein, PLK1 (polo-like kinase 1), in HCC cells. HCC xenograft mouse models revealed that the daily oral administration of HO-1089 retarded tumor growth without systemic toxicity in vivo. The use of HO-1197, a novel herbal formula derived from HO-1089, resulted in statistically significant improved anticancer efficacy relative to HO-1089 in HCC. These results suggest that HO-1089 is a safe and potent integrated natural medicine for HCC therapy

Patient-derived multicellular tumor spheroids towards optimized treatment for patients with hepatocellular carcinoma

Abstract Background Hepatocellular carcinoma (HCC) is one of the most common malignant tumors worldwide and has poor prognosis. Specially, patients with HCC usually have poor tolerance of systemic chemotherapy, because HCCs develop from chronically damaged tissue that contains considerable inflammation, fibrosis, and cirrhosis. Since HCC exhibits highly heterogeneous molecular characteristics, a proper in vitro system is required for the study of HCC pathogenesis. To this end, we have established two new hepatitis B virus (HBV) DNA-secreting HCC cell lines from infected patients. Methods Based on these two new HCC cell lines, we have developed chemosensitivity assays for patient-derived multicellular tumor spheroids (MCTSs) in order to select optimized anti-cancer drugs to provide more informative data for clinical drug application. To monitor the effect of the interaction of cancer cells and stromal cells in MCTS, we used a 3D co-culture model with patient-derived HCC cells and stromal cells from human hepatic stellate cells, human fibroblasts, and human umbilical vein endothelial cells to facilitate screening for optimized cancer therapy. Results To validate our system, we performed a comparison of chemosensitivity of the three culture systems, which are monolayer culture system, tumor spheroids, and MCTSs of patient-derived cells, to sorafenib, 5-fluorouracil, and cisplatin, as these compounds are typically standard therapy for advanced HCC in South Korea. Conclusion In summary, these findings suggest that the MCTS culture system is the best methodology for screening for optimized treatment for each patients with HCC, because tumor spheroids not only mirror the 3D cellular context of the tumors but also exhibit therapeutically relevant pathophysiological gradients and heterogeneity of in vivo tumors

Anti-Cancer Effects of YAP Inhibitor (CA3) in Combination with Sorafenib against Hepatocellular Carcinoma (HCC) in Patient-Derived Multicellular Tumor Spheroid Models (MCTS)

Purpose: To assess the expression levels of YAP and TAZ in patient-derived HCC tissue and identify the effects of YAP/TAZ inhibition depending on the baseline YAP/TAZ expression when combined with sorafenib using a patient-derived multicellular tumor spheroid (MCTS) model. Methods: Primary HCC cell lines were established from patient-derived tissue. Six patient-derived HCC cell lines were selected according to YAP/TAZ expression on Western blot: high, medium, low. Then, MCTS was generated by mixing patient-derived HCC cells and stroma cells (LX2, WI38, and HUVECs) and YAP/TAZ expression was assessed using Western blot. Cell viability of MCTS upon 48 h of drug treatment (sorafenib, sorafenib with CA3 0.1 µM, and CA3 (novel YAP1 inhibitor)) was analyzed. Results: Out of six patient-derived HCC cell lines, cell lines with high YAP/TAZ expression at the MCTS level responded more sensitively to the combination therapy (Sorafenib + CA3 0.1 μM) despite the potent cytotoxic effect of CA3 exhibited in all of the patient-derived HCCs. Conclusion: Targeting YAP/TAZ inhibition using the novel YAP1 inhibitor CA3 could be a promising therapeutic strategy to enhance sensitivity to sorafenib especially in HCCs with high YAP/TAZ expression in MCTS

Anti-Cancer Activity of Buthus occitanus Venom on Hepatocellular Carcinoma in 3D Cell Culture

Hepatocellular carcinoma (HCC) is the most dominant primary liver cancer, which can be caused by chronic hepatitis virus infections and other environmental factors. Resection, liver transplantation, and local ablation are only a few of the highly effective and curative procedures presently accessible. However, other complementary treatments can reduce cancer treatment side effects. In this present work, we evaluated the activity of Moroccan scorpion venom Buthus occitanus and its fractions obtained by chromatography gel filtration against HCC cells using a 3D cell culture model. The venom was fractionated by gel filtration chromatography, each fraction and the crude venom was tested on normal hepatocytes (Fa2N-4 cells). Additionally, the fractions and the crude venom were tested on MCTSs (multicellular tumor spheroids), and this latter was generated by cultivate Huh7.5 cancer cell line with WI38 cells, LX2 cells, and human endothelial cells (HUVEC). Our results indicate that Buthus occitanus venom toxin has no cytotoxic effects on normal hepatocytes. Moreover, it is reported that F3 fraction could significantly inhibit the MCTS cells. Other Protein Separation Techniques (High-performance liquid chromatography) are needed in order to identify the most active molecule

What Is the Relationship between Antioxidant Efficacy, Functional Composition, and Genetic Characteristics in Comparing Soybean Resources by Year?

The aim of this study was to analyze the physiological activity of 48 soybean resources harvested in 2020 to identify the soybean resources’ relationships with individual isoflavone compounds and their genetic properties. These data will subsequently be compared with the research results on soybeans harvested in 2019. Initially, with respect to the physiological activity (6 types) and substances (19 types), this study evaluated the differences between the cultivation year (two years), seed coat color (three colors), and the interaction of the year and seed coat color of soybeans through ANOVA. Among the physiological activities, there were differences in the estrogen, estrogen receptor alpha, and UCP-1 (uncoupling protein-1) activities depending on the cultivation year. Moreover, there were differences in NO (nitric oxide), revealing differences in the ABTS (2, 2′-azino-bis-3ethylbenzo-thiazoline-6-sulfonic acid) and DPPH (2, 2-diphenyl-2-picrylhydrazyl) radical scavenging activities due to the seed coat color and the interaction of the year and seed coat color. Soybeans harvested in 2020 exhibited increased ABTS, DPPH, and NO inhibitory activities and reduced estrogen, estrogen receptor alpha, and UCP-1 activities compared to those harvested in 2019. According to the ANOVA results, eight of the nineteen individual derivatives illustrated yearly differences, while three derivatives displayed differences due to the seed coat color. Secondly, according to the relationship between the efficacy, derivative substances, and genetic properties, it was determined that genistein 7-O-(2″-O-apiosyl)glucoside (F5) is the individual isoflavone derivative that affected the six types of physiological activity, on which the genome-wide association study (GWAS) showed no significant differences for genetic properties. These results were inconsistent with the 2019 data, where three types of individual compounds, including F5, were proposed as substances that correlated with efficacy and there was a high correlation with genetic properties. Therefore, this study selected B17, B23, B15, B24, and Y7 as excellent varieties that are stable and highly functional in the cultivation environment, producing only small annual differences. The results of this study will be utilized as basic data for predicting soybean varieties and their cultivation, which have high environmental stability under climate variation and properly retain the functional substances and efficacy