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Mitochondrial DNA (mtDNA) mutations are often observed in various cancer types. Although the correlation between mitochondrial dysfunction and cancer malignancy has been demonstrated by several studies, further research is required to elucidate the molecular mechanisms underlying accelerated tumor development and progression due to mitochondrial mutations. We generated an mtDNA-depleted cell line, ρ⁰, via long-term ethidium bromide treatment to define the molecular mechanisms of tumor malignancy induced by mitochondrial dysfunction. Mitochondrial dysfunction in ρ⁰ cells reduced drug-induced cell death and decreased the expression of pro-apoptotic proteins including p53. The p53 expression was reduced by activation of nuclear factor-κB that depended on elevated levels of free calcium in HCT116/ρ⁰ cells. Overall, these data provide a novel mechanism for tumor development and drug resistance due to mitochondrial dysfunction. [BMB Reports 2018; 51(6): 296-301].

Mitochondria are major organelles that play various roles in cells, and mitochondrial dysfunction is the main cause of numerous diseases. Mitochondrial dysfunction also occurs in many cancer cells, and these changes are known to affect malignancy. The mitochondria of normal embryonic stem cells (ESCs) exist in an undifferentiated state and do not function properly. We hypothesized that mitochondrial dysfunction in cancer cells caused by the depletion of mitochondrial DNA might be similar to the mitochondrial state of ESCs. We generated mitochondria dysfunctional (ρ0) cells from the Hep3B hepatocellular carcinoma cell line and tested whether these ρ0 cells show cancer stem-like properties, such as self-renewal, chemotherapy resistance, and angiogenesis. Compared with Hep3B cells, the characteristics of each cancer stem-like cell were increased in Hep3B/ρ0 cells. The Hep3B/ρ0 cells formed a continuous and large sphere from a single cell. Additionally, the Hep3B/ρ0 cells showed resistance to the anticancer drug doxorubicin because of the increased expression of ATP-binding cassette Subfamily B Member 1. The Hep3B/ρ0 conditioned medium induced more and thicker blood vessels and increased the mobility and invasiveness of the blood vessel cells. Therefore, our data suggest that mitochondrial dysfunction can transform cancer cells into cancer stem-like cells.

Cancer stem cells (CSCs) are a small population of cancer cells that possess characteristics associated with normal stem cells such as self-renewal and differentiation into multiple cell types. Additionally, CSCs have been reported to relationship on tumor relapse after cancer therapy. Mitochondrial dysfunction (MD) has also been known to be associated with cancer malignancy. In this report, the relation of MD and CSCs were initially investigated using mitochondrial-depleted rho0 cells derived from Hep3B hepatocellular carcinoma cell line. The Hep3B/rho0 cells showed cancer stem-like phenotypes including tumor initiation, tumor malignancy, stem cell-related signaling, chemotherapies and radiotherapy resistance. These results suggest that mitochondrial dysfunction induces cancer stem-like phenotypes. Taken together, mitochondrial dysfunction plays an important role in the acquisition of cancer stem-like phenotypes and could be a novel therapeutic target to cancer therapy. Citation Format: Yu-Seon Han, Eui-Yeun Yi, Yung-Jin Kim. Mitochondrial dysfunction induces cancer stem-like phenotypes in Hep3B hepatocellular carcinoma cell line. [abstract]. In: Proceedings of the AACR Special Conference on Translation of the Cancer Genome; Feb 7-9, 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 1):Abstract nr A2-22.

Hepatitis B virus-encoded HBx is well known causative factor for the Hepatocellular carcinoma (HCC). HBx interferes with several signaling pathways that are associated with cell proliferation and apoptosis. HBx c-terminal region has been known to play important roles in HBV associated HCC. HBx C-terminal mutant was known to show increased proliferation and invasiveness. In this study, we investigated a molecular mechanism of cancer malignancy induced by C-terminal truncated HBx. Huh-7 Hepatocellular carcinoma cells stably transfected with C-terminal truncated HBx showed higher migrative and invasive phenotype. In addition, the cells were more resistant to doxorubicin compared to cells transfected with full-length HBx. We also observed a higher efflux activity in the cells transfected with C-terminal truncated HBx due to the increased expression of ABCB1. These results suggest that C-terminal truncated HBx up-regulate ABCB1 expression which results in the enhanced efflux of drug. Therefore cancer cells containing C-terminal truncated HBx acquire drug resistancy, and show more malignant phenotypes. Citation Format: Myeong-Eun Je Gal, Eui-Yeun Yi, Seung-Youn Jung, Yung-Jin Kim. Cancer malignancy induced by C-terminal truncated HBx in Huh-7 Hepatocellular carcinoma cells. [abstract]. In: Proceedings of the AACR Special Conference on Translation of the Cancer Genome; Feb 7-9, 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 1):Abstract nr A2-21.

Mitochondrial dysfunction has been found to be associated with various pathological conditions, particularly cancer. However, the mechanisms underlying tumor malignancy induced by mitochondrial dysfunction are not fully understood. In the present study, the effects of mitochondrial dysfunction on epithelial-mesenchymal transition (EMT), were investigated using mitochondrial-depleted ρ(0) cells derived from the Hep3B hepatocarcinoma cell line. The Hep3B/ρ(0) cells displayed the EMT phenotype with more aggressive migration and higher invasiveness compared to their parental cells. The Hep3B/ρ(0) cells also showed typical expression pattern of EMT markers such as vimentin and E-cadherin. These phenotypes in Hep3B/ρ(0) cells were mediated by increased transforming growth factor-β (TGF-β) through the canonical Smad-dependent signaling pathway. Additionally, TGF-β signaling was activated via induction of c-Jun/AP-1 expression and activity. Therefore, mitochondrial dysfunction induces EMT through TGF-β/Smad/Snail signaling via c-Jun/AP-1 activation. These results indicate that mitochondrial dysfunction plays an important role in the EMT process and could be a novel therapeutic target for malignant cancer therapy.

Angiogenesis is an important process involved in tumor growth and metastasis. Many studies have investigated the use of natural compounds such as angiogenic inhibitors. Xylitol is a 5-carbon sugar alcohol and is an artificial sweetener that has been used in chewing gums to prevent tooth decay. Xylitol has been also known to inhibit inflammatory cytokine expression induced by lipopolysaccharide (LPS). Since angiogenesis and inflammation share a common signaling pathway, we investigated the role of xylitol in angiogenesis. Xylitol inhibited the migration, invasion and tube formation of human umbilical vein endothelial cells (HUVECs). Xylitol also inhibited in vivo angiogenesis in a mouse Matrigel plug assay. Furthermore, mRNA expression of vascular endothelial growth factor (VEGF), VEGFR-II (KDR), basic fibroblast growth factor (bFGF), bFGFR-II, matrix metalloproteinase-2 (MMP-2) and MMP-9 of HUVECs decreased following treatment with xylitol. These anti-angiogenic effects of xylitol are exerted through inhibition of NF-κB and Akt activation. Taken together, these results suggest that xylitol acts as a beneficial angiogenesis inhibitor.

Ethyl pyruvate (EP), simple derivative of pyruvate, has been shown to have anti-inflammatory properties. Here, we demonstrate EP's strong anti-angiogenic activity. EP inhibited in vivo angiogenesis in the mouse Matrigel-plug assay and tumor growth in the mouse Lewis lung carcinoma model. EP also interfered with the angiogenic cascade, including growth, invasion, migration, and tube formation. Activation of NF-κB by vascular endothelial cell growth factor was reduced by EP. Taken together, we suggest that EP may have potential as a new multi-functional drug candidate for anti-angiogenesis and cancer therapy.

Angiogenesis is considered to be an integral process to the growth and spread of solid tumors. Anti-angiogenesis therapy recently has been found to be one of the most promising anti-cancer therapeutic strategies. In this study, we provide several lines of evidences showing that KR-31831, a new benzopyran derivative, has anti-angiogenic activities. KR-31831 inhibited the proliferation, migration, invasion and tube formation of bovine aortic endothelial cells (BAECs), and suppressed the release of matrix metalloproteinase-2 (MMP-2) of BAECs. KR-31831 also inhibited in vivo angiogenesis in mouse Matrigel plug assay. Furthermore, the mRNA expressions of basic fibroblast growth factor (bFGF), fibroblast growth factor receptor-2 (FGFR-2), and vascular endothelial growth factor receptor-2 (VEGFR-2) were decreased by KR-31831. Taken together, these results suggest that KR-31831 acts as a novel angiogenesis inhibitor and might be useful for treating hypervascularized cancers.

In order to investigate the anti-angiogenic activity of shark TIMP-3 (sTIMP-3) in endothelial cells, angiogenic assays including in vitro invasion assay, migration assay, zymogram assay and tube formation assay were performed. We observed that the overexpression of sTIMP-3 decreased the invasive capacity by about 70%, the migratory activity by about 50% and the production of gelatinase A in bovine aortic endothelial cells (BAECs). In addition, the overexpression of sTIMP-3 interfered with the formation of capillary-like network in endothelial cells. We also examined whether sTIMP-3 shows the anti-invasive activity in cancer cells. We found that the overexpression of sTIMP-3 diminished the invasive ability of the human fibrosarcoma HT1080 cells by about 40%. Also, the production of specific gelatinases was suppressed in the cancer cells. Therefore, we propose that sTIMP-3 acts as the inhibitor of angiogenesis in endothelial cells and the suppressor of tumor invasion in human fibrosarcoma HT1080 cells.

Coenzyme Q10 (CoQ10) is an essential factor of the mitochondrial respiratory chain and has effective antioxidant properties. Therefore, CoQ10 has been used in a variety of clinical applications and used as a nutritional supplement to treat several human diseases. Here, we tested the effects of CoQ10 on angiogenesis stimulated by basic fibroblast growth factor (bFGF). CoQ10 significantly inhibited bFGF-induced angiogenesis in a mouse Matrigel plug and the sprouting of endothelial cells in rat aortic rings. In addition, CoQ10 decreased the ability of tube formation, migration, and invasion in endothelial cells. When CoQ10 was used to inhibit angiogenesis in endothelial cells, the expression of vascular endothelial growth factor (VEGF) and the phosphorylation of ERK were decreased. Taken together, these results indicate that CoQ10 is able to act as an antiangiogenic regulator, and its inhibitory activity is mediated by blocking an ERK-dependent pathway. This study suggests that CoQ10 may be used a therapeutic agent to decrease neovascularization in several diseases, including solid tumors.

Angiogenesis is defined as the formation of new blood vessels form existing vessels surrounding a tumor. The process of angiogenesis is an important step for tumor growth and metastasis, as is inflammation. Thus, angiogenesis inhibitors that suppress inflammation have been studied as an anticancer treatment. Recently, many research groups have investigated the anti-angiogenic activity of natural compounds since some have been demonstrated to have anticancer properties. Among many natural compounds, we focused on betaine, which is known to suppress inflammation. Betaine, trimethylglycine (TMG), was first discovered in the juice of sugar beets and was later shown to be present in wheat, shellfish and spinach. In Southeast Asia, betaine is used in traditional oriental medicine for the treatment of hepatic disorders. Here, we report the anti-angiogenic action of betaine. Betaine inhibited in vitro angiogenic cascade, tube formation, migration and invasion of human umbilical vein endothelial cells (HUVECs). Betaine also inhibited in vivo angiogenesis in the mouse Matrigel plug assay. The mRNA expression levels of basic fibroblast growth factor (bFGF), matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) in HUVECs were decreased by betaine treatment. In addition, betaine suppressed NF-κB and Akt activation.

Angiogenesis is an important mediator of tumor progression. As tumors expand, diffusion distances from the existing vascular supply increases, resulting in hypoxia in the cancer cells. Sustained expansion of a tumor mass requires new blood vessel formation to provide rapidly proliferating tumor cells with an adequate supply of oxygen and nutrients. The key regulator of hypoxia-induced angiogenesis is the transcription factor known as hypoxia-inducible factor (HIF)-1. HIF-1alpha is stabilized by hypoxia-induced reactive oxygen species (ROS) and enhances the expression of several types of hypoxic genes, including that of the angiogenic activator known as vascular endothelial cell growth factor (VEGF). In this study, we found that melatonin, a small lipophilic molecule secreted primarily by the pineal gland, destabilizes hypoxia-induced HIF-1alpha protein levels in the HCT116 human colon cancer cell line. This destabilization of HIF-1alpha resulted from the antioxidant activity of melatonin against ROS induced by hypoxia. Moreover, under hypoxia, melatonin suppressed HIF-1 transcriptional activity, leading to a decrease in VEGF expression. Melatonin also blocked in vitro tube formation and invasion and migration of human umbilical vein endothelial cells induced by hypoxia-stimulated conditioned media of HCT116 cells. These findings suggest that melatonin could play a pivotal role in tumor suppression via inhibition of HIF-1-mediated angiogenesis.

Mitochondria dysfunctional cells are useful models in studies for mitochondrial diseases, apoptosis, cancer and aging, but their functional aspects have not been fully understood. Mitochondrial dysfunction (MD) is found in many type of cancer such as colorectal, liver, pancreatic, breast, prostate, ovarian cancer etc. MD has also been known to be associated with cancer malignancy, especially apoptosis resistance (therapy resistance). Using mitochondrial-depleted P0 derived from Hep3B, we have tested the effects of mitochondrial dysfunction on the chemical and radiation resistance. When compared to parental cells, Hep3B/P0 cells were much less sensitive to doxorubicin and sorafenib and irradiation. Hep3B/P0 cells also showed increased efflux, decreased target molecules, rapid repair and DNA protection. These results suggest that mitochondrial dysfunction confers therapy resistance on the cancer cells, which could be a novel therapeutic target for cancer therapy. Citation Format: YuSeon Han, MyeongEun Je Gal, EuiYeun Yi, YungJin Kim. Mitochondrial dysfunction confers therapy resistance in Hep3B hepatocellular carcinoma cell line. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C67.

A new piperazine derivative, SJ-8026, is a synthetic anti-cancer agent which exhibits topoisomerase II-inhibiting activities. In this study, we investigated the possibility that this compound inhibits angiogenesis and induces tumor-cell apoptosis using bovine aortic endothelial cells (BAECs) and human hepatocellular carcinoma cells (HepG2) as a model system. in vivo, SJ-8026 decreased the neovascularization of chick embryos and the basic fibroblast growth factor-induced angiogenesis in the chorioallantoic membrane and the mouse Matrigel implants. in vitro, SJ-8026 treatment resulted in the inhibition of proliferation, migration, invasion and tube formation in BAECs. In addition, the treatment of SJ-8026 in HepG2 cells reduced the cell viability, and caused the production of fragmented DNA and the morphological changes corresponding to apoptosis including condensed and fragmented DNA. SJ-8026 also elicited the release of cytochrome c and the activation of caspase-3. Therefore, it is possible that SJ-8026 functions as both angiogenesis inhibitor and apoptosis inducer. Taken together, these results suggest that SJ-8026 may be a candidate for strong anti-cancer agent with the ability to inhibit the angiogenesis of endothelial cells and to induce the apoptosis of tumor cells.

A new piperazine derivative, SJ-8002, is a synthetic anti-cancer agent which exhibits microtubule-inhibiting activities. In this study, we investigated the possibility that this compound inhibits angiogenesis and induces tumor-cell apoptosis using bovine aortic endothelial cells (BAECs) and human hepatocellular carcinoma cells (HepG2) as a model system, respectively. In vivo, SJ-8002 decreased the neovascularization of chick embryos and the basic fibroblast growth factor (bFGF)-induced angiogenesis in the chorioallantoic membrane (CAM) and the mouse Matrigel implants, respectively. In vitro, SJ-8002 treatment resulted in the inhibition of proliferation, migration, invasion and tube formation, and of matrix metalloproteinase-2 (MMP-2) expression in BAECs. In addition, the SJ-8002 treatment in HepG2 cells reduced cell viability, and caused the production of fragmented DNA and the morphological changes corresponding to apoptosis including condensed and fragmented DNA in a concentration-dependent manner. SJ-8002 also elicited the release of cytochrome c and the activation of caspase-3. Therefore, it is possible that SJ-8002 functions as both angiogenesis inhibitor and apoptosis inducer. Taken together, these results suggest that SJ-8002 may be a candidate for strong anti-cancer agent with the ability to inhibit the angiogenesis of endothelial cells and to induce the apoptosis of tumor cells.