α-Mangostin attenuates stemness and enhances cisplatin-induced cell death in cervical cancer stem-like cells through induction of mitochondrial-mediated apoptosis

Cancer stem cells (CSCs) exhibit specific characteristics including decontrolled self-renewal, tumor-initiating, promoting, and metastatic potential, abnormal stemness signaling, and chemotherapy resistance. Thus, targeting CSC is becoming an emerging cancer treatment. α-Mangostin has been shown to have potent and multiple anticancer activities. Accordingly, we hypothesized that α-mangostin may diminish the stemness and proliferation of CSC-like cervical cancer cells. In our results, comparing to the parent cells, CSC-like SiHa and HeLa cells highly expressed CSC marker Sox2, Oct4, Nanog, CK-17, and CD49f. α-Mangostin significantly reduced the cell viability, sphere-forming ability, and expression of the CSC stemness makers of CSC-like cervical cancer cells. Further investigation showed that α-mangostin induced mitochondrial depolarization and mitochondrial apoptosis signaling, including upregulation of Bax, downregulation of Mcl-1 and Bcl-2, and activation of caspase-9/3. Moreover, α-mangostin synergically enhanced the cytotoxicity of cisplatin on CSC-like SiHa cells by promoting mitochondrial apoptosis and inhibiting the expression of CSC markers. Consistent with in vitro findings, in vivo tumor growth assay revealed that α-mangostin administration significantly inhibited the growth of inoculated CSC-like SiHa cells and synergically enhanced the antitumor effect of cisplatin. Our findings indicate that α-mangostin can reduce the stemness and proliferation of CSC-like SiHa and HeLa cells and promote the cytotoxicity of cisplatin, which may attribute to the mitochondrial apoptosis activation. Thus, it suggests that α-mangostin may have clinical potential to improve chemotherapy for cervical cancer by targeting cervical CSC.     

SIRT3 Enhances Glycolysis and Proliferation in SIRT3-Expressing Gastric Cancer Cells

SIRT3 is a key NAD⁺-dependent protein deacetylase in the mitochondria of mammalian cells, functioning to prevent cell aging and transformation via regulation of mitochondrial metabolic homeostasis. However, SIRT3 is also found to express in some human tumors; its role in these SIRT3-expressing tumor cells needs to be elucidated. This study demonstrated that the expression of SIRT3 was elevated in a group of gastric cancer cells compared to normal gastric epithelial cells. Although SIRT3 expression levels were increased in the gastric tumor tissues compared to the adjacent non-tumor tissues, SIRT3 positive cancer cells were more frequently detected in the intestinal type gastric cancers than the diffuse type gastric cancers, indicating that SIRT3 is linked with subtypes of gastric cancer. Overexpression of SIRT3 promoted cell proliferation and enhanced ATP generation, glucose uptake, glycogen formation, MnSOD activity and lactate production, which were inhibited by SIRT3 knockdown, indicating that SIRT3 plays a role in reprogramming the bioenergetics in gastric tumor cells. Further analysis revealed that SIRT3 interacted with and deacetylated the lactate dehydrogenase A (LDHA), a key protein in regulating anaerobic glycolysis, enhancing LDHA activity. In consistence, a cluster of glycolysis-associated genes was upregulated in the SIRT3-overexpressing gastric tumor cells. Thus, in addition to the well-documented SIRT3-mediated mitochondrial homeostasis in normal cells, SIRT3 may enhance glycolysis and cell proliferation in SIRT3-expressing cancer cells.     

Inhibition of Aurora-A kinase induces cell cycle arrest in epithelial ovarian cancer stem cells by affecting NFκB pathway

Recurrent ovarian cancer is resistant to conventional chemotherapy. A sub-population of ovarian cancer cells, the epithelial ovarian cancer stem cells (EOC stem cells) have stemness properties, constitutive NFκB activity, and represent the chemoresistant population. Currently, there is no effective treatment that targets these cells. Aurora-A kinase (Aurora-A) is associated with tumor initiation and progression and is overexpressed in numerous malignancies. The aim of this study is to determine the effect of Aurora-A inhibition in EOC stem cells. EOC stem cells were treated with the Aurora-A inhibitor, MK-5108. Cell growth was monitored by Incucyte real-time imaging system, cell viability was measured using the Celltiter 96 assay and cytokine levels were quantified using xMAP technology. The intracellular changes associated with MK-5108 treatment are: (1) polyploidy and cell cycle arrest; (2) inhibition of NFκB activity; (3) decreased cytokine production; and (4) nuclear accumulation of IκBα. Thus, inhibition of Aurora-A decreases cell proliferation in the EOC stem cells by inducing cell cycle arrest and affecting the NFκB pathway. As EOC stem cells represent a source of recurrence and chemoresistance, these results suggest that Aurora-A inhibition may effectively target the cancer stem cell population in ovarian cancer.Key words: ovarian cancer stem cells, aurora-A kinase, cell cycle arrest, nuclear factor kappaB     

Lysine demethylase LSD1 delivered via small extracellular vesicles promotes gastric cancer cell stemness

Several studies have examined the functions of nucleic acids in small extracellular vesicles (sEVs). However, much less is known about the protein cargos of sEVs and their functions in recipient cells. This study demonstrates the presence of lysine‐specific demethylase 1 (LSD1), which is the first identified histone demethylase, in the culture medium of gastric cancer cells. We show that sEVs derived from gastric cancer cells and the plasma of patients with gastric cancer harbor LSD1. The shuttling of LSD1‐containing sEVs from donor cells to recipient gastric cancer cells promotes cancer cell stemness by positively regulating the expression of Nanog, OCT4, SOX2, and CD44. Additionally, sEV‐delivered LSD1 suppresses oxaliplatin response of recipient cells in vitro and in vivo, whereas LSD1‐depleted sEVs do not. Taken together, we demonstrate that LSD1‐loaded sEVs can promote stemness and chemoresistance to oxaliplatin. These findings suggest that the LSD1 content of sEV could serve as a biomarker to predict oxaliplatin response in gastric cancer patients.     

SIRT1 coordinates with the CRL4B complex to regulate pancreatic cancer stem cells to promote tumorigenesis

Pancreatic cancer is a common malignant tumor with poor prognosis. Recently, cancer stem cells (CSCs) were identified in several solid tumors, including pancreatic cancer. Although accumulating evidence indicates that sirtuin 1 (SIRT1) exerts biological functions in various cancers, how it contributes to tumorigenesis and metastasis of pancreatic cancer, as well as its role in CSCs, is still poorly defined. Here we show that SIRT1 interacts with the Cullin 4B (CUL4B)-Ring E3 ligase (CRL4B) complex, which is responsible for H2AK119 monoubiquitination (H2AK119ub1), collaborating as a functional unit. Genome-wide analysis of SIRT1/CUL4B targets identified a cohort of genes, including GRHL3 and FOXO3, critically involved in cell differentiation, growth, and migration. Furthermore, we found that SIRT1 and CUL4B collectively promote the proliferation, autophagy, and invasion of pancreatic cancer cells. Remarkably, we demonstrate that SIRT1/CUL4B promotes CSC-like properties, including increased stemness marker expression and sphere formation. In vivo experiments implied that SIRT1 promoted established tumor xenograft growth, increased tumor-initiating capacity in NOD/SCID mice, and increased CSC frequency. Strikingly, SIRT1 and CUL4B expression is markedly upregulated in a variety of human cancers, including pancreatic cancer. Our data provide a molecular basis for the functional interplay between histone deacetylation and ubiquitination. The results also implicate the SIRT1/CRL4B complex in pancreatic cancer metastasis and stem cell properties, thus supporting SIRT1 as a promising potential target for cancer therapy development.Subject terms: Cancer stem cells, Metastasis     

Metabolic alterations in highly tumorigenic glioblastoma cells: preference for hypoxia and high dependency on glycolysis

Recent studies suggest that a small subpopulation of malignant cells with stem-like properties is resistant to chemotherapy and may be responsible for the existence of residual cancer after treatment. We have isolated highly tumorigenic cancer cells with 100-fold increase in tumor initiating capacity from the tumor xenografts of human glioblastoma U87 cells in mice. These cells exhibit stem-like properties and show unique energy metabolic characteristics including low mitochondrial respiration, increased glycolysis for ATP generation, and preference for hypoxia to maintain their stemness and tumor forming capacity. Mechanistically, mitochondrial depression in the highly tumorigenic cells occurs mainly at complex II of the electron transport chain with a down-regulation of the succinate dehydrogenase subunit B, leading to deregulation of hypoxia-inducible factors. Under hypoxia, the stem-like cancer cells are resistant to conventional anticancer agents but are sensitive to glycolytic inhibition. Furthermore, combination of glycolytic inhibition with standard therapeutic agents is effective in killing the tumor-initiating cells in vitro and inhibits tumor formation in vivo. Our study suggests that stem-like cancer cells prefer a low oxygen microenvironment and actively utilize the glycolytic pathway for ATP generation. Inhibition of glycolysis may be an effective strategy to eradicate residual cancer stem cells that are otherwise resistant to chemotherapeutic agents in their hypoxic niches.     

Melatonin regulates cancer migration and stemness and enhances the anti-tumour effect of cisplatin

Melatonin, a lipophilic hormone released from the pineal gland, has oncostatic effects on various types of cancers. However, its cancer treatment potential needs to be improved by deciphering its corresponding mechanisms of action and optimising therapeutic strategy. In the present study, melatonin inhibited gastric cancer cell migration and soft agar colony formation. Magnetic-activated cell sorting was applied to isolate CD133⁺ cancer stem cells. Gene expression analysis showed that melatonin lowered the upregulation of LC3-II expression in CD133⁺ cells compared to CD133⁻ cells. Several long non-coding RNAs and many components in the canonical Wnt signalling pathway were altered in melatonin-treated cells. In addition, knockdown of long non-coding RNA H19 enhanced the expression of pro-apoptotic genes, Bax and Bak, induced by melatonin treatment. Combinatorial treatment with melatonin and cisplatin was investigated to improve the applicability of melatonin as an anticancer therapy. Combinatorial treatment increased the apoptosis rate and induced G0/G1 cell cycle arrest. Melatonin can regulate migration and stemness in gastric cancer cells by modifying many signalling pathways. Combinatorial treatment with melatonin and cisplatin has the potential to improve the therapeutic efficacy of both.     

Loss of SIRT3 Provides Growth Advantage for B Cell Malignancies

B cell malignancies comprise a diverse group of cancers that proliferate in lymph nodes, bone marrow, and peripheral blood. SIRT3 (sirtuin 3) is the major deacetylase within the mitochondrial matrix that promotes aerobic metabolism and controls reactive oxygen species (ROS) by deacetylating and activating isocitrate dehydrogenase 2 (IDH2) and superoxide dismutase 2 (SOD2). There is controversy as to whether SIRT3 acts as an oncogene or a tumor suppressor, and here we investigated its role in B cell malignancies. In mantle cell lymphoma patient samples, we found that lower SIRT3 protein expression was associated with worse overall survival. Further, SIRT3 protein expression was reduced in chronic lymphocytic leukemia primary samples and malignant B cell lines compared to primary B cells from healthy donors. This lower level of expression correlated with hyperacetylation of IDH2 and SOD2 mitochondrial proteins, lowered enzymatic activities, and higher ROS levels. Overexpression of SIRT3 decreased proliferation and diminished the Warburg-like phenotype in SIRT3-deficient cell lines, and this effect is largely dependent on deacetylation of IDH2 and SOD2. Lastly, depletion of SIRT3 from malignant B cell lines resulted in greater susceptibility to treatment with an ROS scavenger but did not result in greater sensitivity to inhibition of the hypoxia-inducible factor-1α pathway, suggesting that loss of SIRT3 increases proliferation via ROS-dependent but hypoxia-inducible factor-1α-independent mechanisms. Our study suggests that SIRT3 acts as a tumor suppressor in B cell malignancies, and activating the SIRT3 pathway might represent a novel therapeutic approach for treating B cell malignancies.     

siRNA targeting midkine inhibits gastric cancer cells growth and induces apoptosis involved caspase-3,8,9 activation and mitochondrial depolarization

Midkine (MK), a heparin-binding growth factor, is expressed highly in various malignant tumors, so it acts as attractive therapeutic target. In the present study, we used siRNA targeting MK to downregulate human MK expression in human gastric cancer cell line BGC823 and SGC7901 so as to determine the advantages of this anticancer therapeutic. The cell proliferation was evaluated by a WST-8 (4-[3-(2-methoxy-4-nitrophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1, 3-benzene disulfonate sodium salt) assay and colony formation assay. Apoptosis was determined by flow cytometer analysis and colorimetric assay. Our results showed that the BGC823 and SGC7901 cell growth were significantly inhibited by knockdown of MK gene. The loss of mitochondrial membrane potential, release of cytochrome c from the mitochondria into cytosol and increased activity of caspase-3, 8 and 9 occurred concomitantly with inhibition of MK gene. These results indicated that siRNA targeting MK gene can inhibit gastric cancer cells growth and induce apoptosis via mitochondrial depolarization and caspase-3 activation. MK siRNA may be a promising novel and potential therapeutic strategy for the treatment of gastric cancers.     

ONECUT2 which is targeted by hsa-miR-15a-5p enhances stemness maintenance of gastric cancer stem cells

Gastric cancer is the third dominating cause of cancer-associated death. MiroRNAs are potential clinical tools for cancer diagnosis and therapy. In this project, we demonstrated significant overexpression of ONECUT2 and down-regulation of hsa-miR-15a-5p in gastric cancer via bioinformatics analysis and in vitro assays. Meanwhile, ONECUT2 expression is related to clinical prognosis in gastric cancer and inversely proportional to the differentiation degree of gastric adenocarcinoma according to immunohistochemistry results. Then, we separated CD133+/CD44+ MKN45 by flow cytometry and found that, compared with parental MKN45, CD133+/CD44+ MKN45 gastric cancer stem cells (GCSCs) had higher levels of ONECUT2 and lower levels of hsa-miR-15a-5p. In addition, we applied both in vivo and ex vivo assays to demonstrate hsa-miR-15a-5p regulates the stemness maintenance, epithelial–mesenchymal transition, and chemosensitivity of GCSCs through targeting ONECUT2. Also, hsa-miR-15a-5p inhibits G0 phase block of GCSCs by regulating ONECUT2/β-catenin signaling pathway. However, this study has provided novel perspective into the dynamic control of cancer stem cells for advanced gastric cancer treatment.     

Metformin Reduces Hepatic Expression of SIRT3, the Mitochondrial Deacetylase Controlling Energy Metabolism

Metformin inhibits ATP production in mitochondria and this may be involved in the anti-hyperglycemic effects of the drug. Sirtuin 3 (SIRT3) is a mitochondrial protein deacetylase that regulates the function of the electron transport chain and maintains basal ATP yield. We hypothesized that metformin treatment could diminish mitochondrial ATP production through downregulation of SIRT3 expression. Glucagon and cAMP induced SIRT3 mRNA in mouse primary hepatocytes. Metformin prevented SIRT3 induction by glucagon. Moreover, metformin downregulated constitutive expression of SIRT3 in primary hepatocytes and in the liver in vivo. Estrogen related receptor alpha (ERRα) mediates regulation of Sirt3 gene by peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α). ERRα mRNA expression was regulated in a similar manner as SIRT3 mRNA by glucagon, cAMP and metformin. However, a higher metformin concentration was required for downregulation of ERRα than SIRT3. ERRα siRNA attenuated PGC-1α mediated induction of SIRT3, but did not affect constitutive expression. Overexpression of the constitutively active form of AMP-activated protein kinase (AMPK) induced SIRT3 mRNA, indicating that the SIRT3 downregulation by metformin is not mediated by AMPK. Metformin reduced the hepatocyte ATP level. This effect was partially counteracted by SIRT3 overexpression. Furthermore, metformin decreased mitochondrial SIRT3 protein levels and this was associated with enhanced acetylation of several mitochondrial proteins. However, metformin increased mitochondrial mass in hepatocytes. Altogether, our results indicate that metformin attenuates mitochondrial expression of SIRT3 and suggest that this mechanism is involved in regulation of energy metabolism by metformin in the liver and may contribute to the therapeutic action of metformin.     

Human colonic fibroblasts regulate stemness and chemotherapy resistance of colon cancer stem cells

There is increasing evidence that cancers are heterogeneous and contain a hierarchical organization consisting of cancer stem cells and their differentiated cell progeny. These cancer stem cells are at the core of the tumor as they represent the clonogenic cells within a tumor. Moreover, these cells are considered to contain selective therapy resistance, which suggests a pivotal role in therapy resistance and tumor relapse. Here we show that differentiated cells can re-acquire stemness through factors secreted from fibroblasts. This induced CSC state also coincides with re-acquisition of resistance to chemotherapy. Resistance induced in newly formed CSCs is mediated by the anti-apoptotic molecule BCLXL and inhibition of BCLXL with the BH3 mimetic ABT-737 sensitizes these cancer cells toward chemotherapy. These data point to an important interplay between tumor cells and their microenvironment in the regulation of stemness and therapy resistance.