Hypoxia induces downregulation of the tumor-suppressive sST2 in colorectal cancer cells via the HIF-nuclear IL-33-GATA3 pathway.

As a decoy receptor, soluble ST2 (sST2) interferes with the function of the inflammatory cytokine interleukin (IL)-33. Decreased sST2 expression in colorectal cancer (CRC) cells promotes tumor growth via IL-33-mediated bioprocesses in the tumor microenvironment. In this study, we discovered that hypoxia reduced sST2 expression in CRC cells and explored the associated molecular mechanisms, including the expression of key regulators of ST2 gene transcription in hypoxic CRC cells. In addition, the effect of the recovery of sST2 expression in hypoxic tumor regions on malignant progression was investigated using mouse CRC cells engineered to express sST2 in response to hypoxia. Our results indicated that hypoxia-dependent increases in nuclear IL-33 interfered with the transactivation activity of GATA3 for ST2 gene transcription. Most importantly, hypoxia-responsive sST2 restoration in hypoxic tumor regions corrected the inflammatory microenvironment and suppressed tumor growth and lung metastasis. These results indicate that strategies targeting sST2 in hypoxic tumor regions could be effective for treating malignant CRC.

Anticancer effect of a pyrrole-imidazole polyamide-triphenylphosphonium conjugate selectively targeting a common mitochondrial DNA cancer risk variant in cervical cancer cells.

Cervical cancer remains a major threat to women's health, especially in countries with limited medical resources, and new drugs are needed to improve patient survival and minimize adverse effects. Here, we examine the effects of a triphenylphosphonium (TPP)-conjugated pyrrole-imidazole polyamide (CCC-h1005) targeting the common homoplasmic mitochondrial DNA (mtDNA) cancer risk variant (ATP6 8860A>G) on the survival of cervical cancer cell lines, cisplatin-resistant HeLa cells and patient-derived cervical clear cell carcinoma cells as models of cervical cancer treatment. We found that CCC-h1005 induced death in these cells and suppressed the growth of xenografted HeLa tumors with no severe adverse effects. These results suggest that PIP-TPP designed to target mtDNA cancer risk variants can be used to treat many cervical cancers harboring high copies of the target variant, providing a foundation for clinical trials of this class of molecules for treating cervical cancer and other types of cancers.

Suppression of non-small-cell lung cancer A549 tumor growth by an mtDNA mutation-targeting pyrrole-imidazole polyamide-triphenylphosphonium and a senolytic drug.

Certain somatic mutations in mtDNA were associated with tumor progression and frequently found in a homoplasmic state. We recently reported that pyrrole-imidazole polyamide conjugated with the mitochondria-delivering moiety triphenylphosphonium (PIP-TPP) targeting an mtDNA mutation efficiently induced apoptosis in cancer cells with the mutation but not normal cells. Here, we synthesized the novel PIP-TPP, CCC-021-TPP, targeting ND6 14582A > G homoplasmic missense mutation that is suggested to enhance metastasis of non-small-cell lung cancer A549 cells. CCC-021-TPP did not induce apoptosis but caused cellular senescence in the cells, accompanied by a significant induction of antiapoptotic BCL-XL. Simultaneous treatment of A549 cells with CCC-021-TPP and the BCL-XL selective inhibitor A-1155463 resulted in apoptosis induction. Importantly, the combination induced apoptosis and suppressed tumor growth in an A549 xenografted model. These results highlight the potential of anticancer therapy with PIP-TPPs targeting mtDNA mutations to induce cell death even in apoptosis-resistant cancer cells when combined with senolytics.

Intercellular transfer of mitochondrial DNA carrying metastasis-enhancing pathogenic mutations from high- to low-metastatic tumor cells and stromal cells via extracellular vesicles.

BACKGROUND: Mitochondrial DNA (mtDNA) carrying certain pathogenic mutations or single nucleotide variants (SNVs) enhances the invasion and metastasis of tumor cells, and some of these mutations are homoplasmic in tumor cells and even in tumor tissues. On the other hand, intercellular transfer of mitochondria and cellular components via extracellular vesicles (EVs) and tunneling nanotubes (TNTs) has recently attracted intense attention in terms of cell-to-cell communication in the tumor microenvironment. It remains unclear whether metastasis-enhancing pathogenic mutant mtDNA in tumor cells is intercellularly transferred between tumor cells and stromal cells. In this study, we investigated whether mtDNA with the NADH dehydrogenase subunit 6 (ND6) G13997A pathogenic mutation in highly metastatic cells can be horizontally transferred to low-metastatic cells and stromal cells in the tumor microenvironment. RESULTS: When MitoTracker Deep Red-labeled high-metastatic Lewis lung carcinoma A11 cells carrying the ND6 G13997A mtDNA mutation were cocultured with CellLight mitochondria-GFP-labeled low-metastatic P29 cells harboring wild-type mtDNA, bidirectional transfer of red- and green-colored vesicles, probably mitochondria-related EVs, was observed in a time-dependent manner. Similarly, intercellular transfer of mitochondria-related EVs occurred between A11 cells and α-smooth muscle actin (α-SMA)-positive cancer-associated fibroblasts (CAFs, WA-mFib), macrophages (RAW264.7) and cytotoxic T cells (CTLL-2). Intercellular transfer was suppressed by inhibitors of EV release. The large and small EV fractions (L-EV and S-EV, respectively) prepared from the conditioned medium by differential ultracentrifugation both were found to contain mtDNA, although only S-EVs were efficiently incorporated into the cells. Several subpopulations had evidence of LC3-II and contained degenerated mitochondrial components in the S-EV fraction, signaling to the existence of autophagy-related S-EVs. Interestingly, the S-EV fraction contained a MitoTracker-positive subpopulation, which was inhibited by the respiration inhibitor antimycin A, indicating the presence of mitochondria with membrane potential. It was also demonstrated that mtDNA was transferred into mtDNA-less ρ0 cells after coculture with the S-EV fraction. In syngeneic mouse subcutaneous tumors formed by a mixture of A11 and P29 cells, the mitochondria-related EVs released from A11 cells reached distantly positioned P29 cells and CAFs. CONCLUSIONS: These results suggest that metastasis-enhancing pathogenic mtDNA derived from metastatic tumor cells is transferred to low-metastatic tumor cells and stromal cells via S-EVs in vitro and in the tumor microenvironment, inferring a novel mechanism of enhancement of metastatic potential during tumor progression.

Mitochondria: Endosymbiont bacteria DNA sequence as a target against cancer.

As the energy factory for the cell, the mitochondrion, through its role of adenosine triphosphate production by oxidative phosphorylation, can be regarded as the guardian of well regulated cellular metabolism; the integrity of mitochondrial functions, however, is particularly vulnerable in cancer due to the lack of superstructures such as histone and lamina folds to protect the mitochondrial genome from unintended exposure, which consequently elevates risks of mutation. In cancer, mechanisms responsible for enforcing quality control surveillance for identifying and eliminating defective mitochondria are often poorly regulated, and certain uneliminated mitochondrial DNA (mtDNA) mutations and polymorphisms can be advantageous for the proliferation, progression, and metastasis of tumor cells. Such pathogenic mtDNA aberrations are likely to increase and occasionally be homoplasmic in cancer cells and, intriguingly, in normal cells in the proximity of tumor microenvironments as well. Distinct characteristics of these abnormalities in mtDNA may provide a new path for cancer therapy. Here we discuss a promising novel therapeutic strategy, using the sequence-specific properties of pyrrole-imidazole polyamide-triphenylphosphonium conjugates, against cancer for clearing abnormal mtDNA by reactivating mitochondrial quality control surveillance.

A linear five-ring pyrrole-imidazole polyamide-triphenylphosphonium conjugate targeting a mitochondrial DNA mutation efficiently induces apoptosis of HeLa cybrid cells carrying the mutation.

Somatic mutations in mitochondrial DNA may provide a new avenue for cancer therapy due to their associations to a number of cancers and a tendency of homoplasmicity. In consideration of mitochondrial features and its relatively small genome size, a nucleotide-based targeting approach is a considerably more promising option. To explore the efficacy of short linear N-methylpyrrole-N-methylimidazole polyamide (PI polyamide), we synthesized a five-ring short PI polyamide that provided sequence-specific homing for the A3243G mitochondrial mutation upon conjugation with triphenylphosphonium cation (TPP). This PI polyamide-TPP was able to induce cytotoxicity in HeLamtA3243G cybrid cells, while preserving preferential binding for oligonucleotides containing the A3243G motif from melting temperature assays. The PI polyamide-TPP also localized in the mitochondria in HeLamtA3243G cells and induced mitochondrial reactive oxygen species production, mitophagy and apoptosis in a mutation-specific fashion compared to the wild-type HeLamtHeLa cybrids; normal human dermal fibroblasts were also relatively unaffected to suggest discriminating selectivity for the mutant mitochondria, offering a novel outlook for cancer therapy via mitochondrial homing of short linear PIP-TPPs.

MCT4 is induced by metastasis-enhancing pathogenic mitochondrial NADH dehydrogenase gene mutations and can be a therapeutic target.

Pathogenic mitochondrial NADH dehydrogenase (ND) gene mutations enhance the invasion and metastasis of various cancer cells, and they are associated with metastasis in human non-small cell lung cancer (NSCLC). Moreover, monocarboxylate transporter 4 (MCT4) is overexpressed in solid cancers and plays a role in cancer cell proliferation and survival. Here, we report that MCT4 is exclusively expressed in mouse transmitochondrial cybrids with metastasis-enhancing pathogenic ND6 mutations. A high level of MCT4 is also detected in human NSCLC cell lines and tissues predicted to carry pathogenic ND mutations and is associated with poor prognosis in NSCLC patients. MCT4 expression in the cell lines is suppressed by N-acetyl-L-cysteine. Phosphatidylinositol-3 kinase (PI3K), AMP-activated protein kinase (AMPK) and mechanistic target of rapamycin (mTOR) are involved in the regulation of MCT4 expression in the transmitochondrial cybrid cells. An MCT1/4 inhibitor effectively kills NSCLC cells with predicted pathogenic ND mutations, but an MCT1/2 inhibitor does not have the same effect. Thus, MCT4 expression is augmented by pathogenic ND mutations and could be a biomarker and a therapeutic target in pathogenic ND mutation-harbouring metastatic tumours.

A PI polyamide-TPP conjugate targeting a mtDNA mutation induces cell death of cancer cells with the mutation.

Mitochondrial DNA (mtDNA) mutations occur frequently in cancer cells, and some of them are often homoplasmic. Targeting such mtDNA mutations could be a new method for killing cancer cells with minimal impact on normal cells. Pyrrole-imidazole polyamides (PIPs) are cell-permeable minor groove binders that show sequence-specific binding to double-stranded DNA and inhibit the transcription of target genes. PIP conjugated with the lipophilic triphenylphosphonium (TPP) cation can be delivered to mitochondria without uptake into the nucleus. Here, we investigated the feasibility of the use of PIP-TPP to target a mtDNA mutation in order to kill cancer cells that harbor the mutation. We synthesized hairpin-type PIP-TPP targeting the A3243G mutation and examined its effects on the survival of HeLa cybrid cells with or without the mutation (HeLamtA3243G cells or HeLamtHeLa cells, respectively). A surface plasmon resonance assay demonstrated that PIP-TPP showed approximately 60-fold higher binding affinity for the mutant G-containing synthetic double-stranded DNA than for the wild-type A-containing DNA. When added to cells, it localized in mitochondria and induced mitochondrial reactive oxygen species production, extensive mitophagy, and apoptosis in HeLamtA3243G cells, while only slightly exerting these effects in HeLamtHeLa cells. These results suggest that PIP-TPPs targeting mtDNA mutations could be potential chemotherapeutic drugs to treat cancers without severe adverse effects.

Inhibition of the invasion and metastasis of mammary carcinoma cells by NBD peptide targeting S100A4 via the suppression of the Sp1/MMP­14 axis.

A synthetic peptide that blocks the interaction between the metastasis­enhancing calcium­binding protein, S100A4, and its effector protein, methionine aminopeptidase 2 (MetAP2) (the NBD peptide), was previously demonstrated to inhibit the angiogenesis of endothelial cells, leading to the regression of human prostate cancer in a xenograft model. However, the effects of the NBD peptide on the malignant properties of cancer cells that express S100A4 remain to be elucidated. The present study demonstrates that the NBD peptide inhibits the invasiveness and metastasis of highly metastatic human mammary carcinoma cells. The introduction of the peptide into MDA­MB­231 variant cells resulted in the suppression of matrix degradation in a gelatin invadopodia assay and invasiveness in a Matrigel invasion assay. In line with these results, the peptide significantly downregulated the expression of matrix metalloproteinase (MMP)­14 (MT1­MMP). Mechanistic analysis of the downregulation of MMP­14 revealed the suppression of the expression of the transcription factor, specificity protein 1 (Sp1), but not that of nuclear factor (NF)­κB, early growth response 1 (EGR1) or ELK3, all of which were reported to be involved in transcriptional regulation of the MMP­14 gene. At the same time, evidence suggested that the NBD peptide also suppressed Sp1 and MMP­14 expression levels in MDA­MB­468 cells. Importantly, the intravenous administration of the NBD peptide encapsulated in liposomes inhibited pulmonary metastasis from mammary gland tumors in mice with xenograft tumors. These results indicate that the NBD peptide can suppress malignant tumor growth through the suppression of the Sp1/MMP­14 axis. Taken together, these results reveal that the NBD peptide acts on not only endothelial cells, but also on tumor cells in an integrated manner, suggesting that the peptide may prove to be a promising cancer therapeutic peptide drug.

Obesity reduces the anticancer effect of AdipoRon against orthotopic pancreatic cancer in diet-induced obese mice.

The antidiabetic adiponectin receptor agonist AdipoRon has been shown to suppress the tumour growth of human pancreatic cancer cells. Because obesity and diabetes affect pancreatic cancer progression and chemoresistance, we investigated the effect of AdipoRon on orthotopic tumour growth of Panc02 pancreatic cancer cells in DIO (diet-induced obese) prediabetic mice. Administration of AdipoRon into DIO mice fed high-fat diets, in which prediabetic conditions were alleviated to some extent, did not reduce either body weight or tumour growth. However, when the DIO mice were fed low-fat diets, body weight and the blood leptin level gradually decreased, and importantly, AdipoRon became effective in suppressing tumour growth, which was accompanied by increases in necrotic areas and decreases in Ki67-positive cells and tumour microvessels. AdipoRon inhibited cell growth and induced necrotic cell death of Panc02 cells and suppressed angiogenesis of endothelial MSS31 cells. Insulin and IGF-1 only slightly reversed the AdipoRon-induced suppression of Panc02 cell survival but had no effect on the AdipoRon-induced suppression of MSS31 cell angiogenesis. Leptin significantly ameliorated AdipoRon-induced suppression of angiogenesis through inhibition of ERK1/2 activation. These results suggest that obesity-associated factors weaken the anticancer effect of AdipoRon, which indicates the importance of weight loss in combating pancreatic cancer.

Cancer cell-derived interleukin-33 decoy receptor sST2 enhances orthotopic tumor growth in a murine pancreatic cancer model.

BACKGROUND: Proinflammatory interleukin-33 (IL-33) binds to its receptor ST2L and is involved in inflammation and the malignant behavior of cancer cells. However, the role of IL-33-ST2L and the IL-33 decoy receptor sST2 in the tumor microenvironment of pancreatic cancer is unclear. Because we previously reported that sST2 derived from colon cancer cells profoundly influences malignant tumor growth, we hypothesized that sST2 released from pancreatic cancer cells also modulates IL-33-ST2L signaling in the tumor microenvironment, thereby influencing tumor growth. METHODS: ST2 (ST2L and sST2) expression in mouse pancreatic cancer Panc02 cells was downregulated by shRNAs. mRNA expression levels of IL-33, ST2, cytokines and chemokines in the cells and tumor tissues were examined using real-time PCR. sST2 secretion and the amount of CXCL3 in tumor tissues were measured using ELISA. Tumor growth was investigated after injection of the cells into the pancreas of C57BL/6 mice. MPO+, F4/80+ and CD20+ cells in tumor tissues were detected using immunohistochemistry. RESULTS: Some but not all human and mouse pancreatic cancer cell lines preferentially expressed sST2. Then, we investigated the role of sST2 in orthotopic tumor growth of sST2-expressing mouse pancreatic cancer Panc02 cells in immunocompetent mice. shRNA-mediated knockdown of sST2 expression in the cells suppressed orthotopic tumor growth, which was partially recovered by overexpression of shRNA-resistant sST2 mRNA but was not evident in IL-33 knockout mice. This was associated with decreases in Cxcl3 expression, vessel density and accumulation of cancer-associated neutrophils but not cancer-associated macrophages. Administration of SB225002, an inhibitor of the CXCL3 receptor CXCR2, induced similar effects. CONCLUSIONS: Cancer cell-derived sST2 enhances tumor growth through upregulation of CXCL3 via inhibition of IL-33-ST2L signaling in the tumor microenvironment of pancreatic cancer. These results suggest that the sST2 and the CXCL3-CXCR2 axis could be therapeutic targets.

Role of the IL-33/ST2L axis in colorectal cancer progression.

Interleukin-33 (IL-33) has been identified as a natural ligand of ST2L. IL-33 primarily acts as a key regulator of Th2 responses through binding to ST2L, which is antagonized by soluble ST2 (sST2). The IL-33/ST2L axis is involved in various inflammatory pathologies, including ulcerative colitis (UC). Several recent investigations have also suggested that the IL-33/ST2L axis plays a role in colorectal cancer (CRC) progression. In CRC, tumor- and stroma-derived IL-33 may activate ST2L on various cell types in an autocrine and paracrine manner. Although several findings support the hypothesis that the IL-33/ST2L axis positively regulates CRC progression, other reports do not; hence, this hypothesis remains controversial. At any rate, recent studies have provided overwhelming evidence that the IL-33/ST2L axis plays important roles in CRC progression. This review summarizes the role of the IL-33/ST2L axis in the UC and CRC microenvironments.

Antidiabetic adiponectin receptor agonist AdipoRon suppresses tumour growth of pancreatic cancer by inducing RIPK1/ERK-dependent necroptosis.

The association between lower circulating adiponectin (APN) levels and the development of pancreatic cancer has been reported. However, the effect of APN on the growth and survival of pancreatic cancer cells remains elusive. Here, we investigate the effects of the anti-diabetic APN receptor (AdipoR) agonist AdipoRon and APN on human pancreatic cancer cells. We found that AdipoRon, but not APN, induces MIAPaCa-2 cell death, mainly through necroptosis. Mechanistically, although both AdipoRon and APN activate AMPK and p38 MAPK in an AdipoR-dependent manner that elicits survival signals, only AdipoRon induces rapid mitochondrial dysfunction through mitochondrial Ca2+ overload, followed by superoxide production via RIPK1 and ERK1/2 activation. Oral administration of AdipoRon suppresses MIAPaCa-2 tumour growth without severe adverse effects and kills cancer cells isolated from patients with pancreatic cancer. Thus, AdipoRon could be a therapeutic agent against pancreatic cancer as well as diabetes.

Propofol induces a metabolic switch to glycolysis and cell death in a mitochondrial electron transport chain-dependent manner.

The intravenous anesthetic propofol (2,6-diisopropylphenol) has been used for the induction and maintenance of anesthesia and sedation in critical patient care. However, the rare but severe complication propofol infusion syndrome (PRIS) can occur, especially in patients receiving high doses of propofol for prolonged periods. In vivo and in vitro evidence suggests that the propofol toxicity is related to the impaired mitochondrial function. However, underlying molecular mechanisms remain unknown. Therefore, we investigated effects of propofol on cell metabolism and death using a series of established cell lines of various origins, including neurons, myocytes, and trans-mitochondrial cybrids, with defined mitochondrial DNA deficits. We demonstrated that supraclinical concentrations of propofol in not less than 50 µM disturbed the mitochondrial function and induced a metabolic switch, from oxidative phosphorylation to glycolysis, by targeting mitochondrial complexes I, II and III. This disturbance in mitochondrial electron transport caused the generation of reactive oxygen species, resulting in apoptosis. We also found that a predisposition to mitochondrial dysfunction, caused by a genetic mutation or pharmacological suppression of the electron transport chain by biguanides such as metformin and phenformin, promoted propofol-induced caspase activation and cell death induced by clinical relevant concentrations of propofol in not more than 25 µM. With further experiments with appropriate in vivo model, it is possible that the processes to constitute the molecular basis of PRIS are identified.

Association of predicted pathogenic mutations in mitochondrial ND genes with distant metastasis in NSCLC and colon cancer.

Cancer cells have more mutations in their mitochondrial DNA (mtDNA) than do normal cells, and pathogenic mutations in the genes encoding mitochondrial NADH dehydrogenase (ND) subunits have been found to enhance the invasive and metastatic ability of various tumour cells in animal experiments. However, it is unknown whether single-nucleotide variants (SNVs) of the ND genes that decrease complex I activity are involved in distant metastasis in human clinical samples. Here, we demonstrated the enhancement of the distant metastasis of Lewis lung carcinoma cells by the ND6 13885insC mutation, which is accompanied by the overexpression of metastasis-related genes, metabolic reprogramming, the enhancement of tumour angiogenesis and the acquisition of resistance to stress-induced cell death. We then sequenced ND genes in primary tumour lesions with or without distant metastases as well as metastatic tumour lesions from 115 patients with non-small cell lung cancer (NSCLC) and colon cancer, and we subsequently selected 14 SNVs with the potential to decrease complex I activity. Intriguingly, a significant correlation was observed (P < 0.05 by Chi-square test) between the incidence of the selected mutations and distant metastasis. Thus, these results strongly suggest that pathogenic ND gene mutations participate in enhancing distant metastasis in human cancers.

Cytoplasmic transfer of heritable elements other than mtDNA from SAMP1 mice into mouse tumor cells suppresses their ability to form tumors in C57BL6 mice.

In a previous study, we generated transmitochondrial P29mtSAMP1 cybrids, which had nuclear DNA from the C57BL6 (referred to as B6) mouse strain-derived P29 tumor cells and mitochondrial DNA (mtDNA) exogenously-transferred from the allogeneic strain SAMP1. Because P29mtSAMP1 cybrids did not form tumors in syngeneic B6 mice, we proposed that allogeneic SAMP1 mtDNA suppressed tumor formation of P29mtSAMP1 cybrids. To test this hypothesis, current study generated P29mt(sp)B6 cybrids carrying all genomes (nuclear DNA and mtDNA) from syngeneic B6 mice by eliminating SAMP1 mtDNA from P29mtSAMP1 cybrids and reintroducing B6 mtDNA. However, the P29mt(sp)B6 cybrids did not form tumors in B6 mice, even though they had no SAMP1 mtDNA, suggesting that SAMP1 mtDNA is not involved in tumor suppression. Then, we examined another possibility of whether SAMP1 mtDNA fragments potentially integrated into the nuclear DNA of P29mtSAMP1 cybrids are responsible for tumor suppression. We generated P29H(sp)B6 cybrids by eliminating nuclear DNA from P29mt(sp)B6 cybrids and reintroducing nuclear DNA with no integrated SAMP1 mtDNA fragment from mtDNA-less P29 cells resistant to hygromycin in selection medium containing hygromycin. However, the P29H(sp)B6 cybrids did not form tumors in B6 mice, even though they carried neither SAMP1 mtDNA nor nuclear DNA with integrated SAMP1 mtDNA fragments. Moreover, overproduction of reactive oxygen species (ROS) and bacterial infection were not involved in tumor suppression. These observations suggest that tumor suppression was caused not by mtDNA with polymorphic mutations or infection of cytozoic bacteria but by hypothetical heritable cytoplasmic elements other than mtDNA from SAMP1 mice.

HIF-1-mediated suppression of mitochondria electron transport chain function confers resistance to lidocaine-induced cell death.

The local anesthetic lidocaine induces cell death by altering reactive oxygen species (ROS) generation and mitochondrial electron transport chain function. Because hypoxia-inducible factor 1 (HIF-1) is involved in determining oxygen metabolism and mitochondria function, we investigated the involvement of HIF-1 activity in lidocaine-induced cell death. We investigated the role of HIF activation on lidocaine-induced caspase activation and cell death in renal cell-derived RCC4 cells lacking functional von Hippel-Lindau (VHL) protein. We demonstrate that HIF-1 suppressed oxygen consumption and facilitated glycolysis in a pyruvate dehydrogenase kinase-1-dependent manner and that activation of HIF-1 conferred resistance to lidocaine-induced cell death. We also demonstrated that exogenous HIF-1 activation, through HIFα-hydroxylase inhibition or exposure to hypoxic conditions, alleviates lidocaine toxicity by suppressing mitochondria function and generating ROS, not only in RCC4 cells, but also in the neuronal SH-SY5Y cells. In conclusion, we demonstrate that HIF-1 activation due to VHL deletion, treatment with small molecule HIFα-hydroxylase inhibitors, and exposure to hypoxic conditions suppresses mitochondrial respiratory chain function and confers resistance to lidocaine toxicity.

HIF-2α dictates the susceptibility of pancreatic cancer cells to TRAIL by regulating survivin expression.

Cancer cells develop resistance to therapy by adapting to hypoxic microenvironments, and hypoxia-inducible factors (HIFs) play crucial roles in this process. We investigated the roles of HIF-1α and HIF-2α in cancer cell death induced by tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) using human pancreatic cancer cell lines. siRNA-mediated knockdown of HIF-2α, but not HIF-1α, increased susceptibility of two pancreatic cancer cell lines, Panc-1 and AsPC-1, to TRAIL in vitro under normoxic and hypoxic conditions. The enhanced sensitivity to TRAIL was also observed in vivo. This in vitro increased TRAIL sensitivity was observed in other three pancreatic cancer cell lines. An array assay of apoptosis-related proteins showed that knockdown of HIF-2α decreased survivin expression. Additionally, survivin promoter activity was decreased in HIF-2α knockdown Panc-1 cells and HIF-2α bound to the hypoxia-responsive element in the survivin promoter region. Conversely, forced expression of the survivin gene in HIF-2α shRNA-expressing Panc-1 cells increased resistance to TRAIL. In a xenograft mouse model, the survivin suppressant YM155 sensitized Panc-1 cells to TRAIL. Collectively, our results indicate that HIF-2α dictates the susceptibility of human pancreatic cancer cell lines, Panc-1 and AsPC-1, to TRAIL by regulating survivin expression transcriptionally, and that survivin could be a promising target to augment the therapeutic efficacy of death receptor-targeting anti-cancer therapy.

Soluble IL-33 receptor sST2 inhibits colorectal cancer malignant growth by modifying the tumour microenvironment.

Interleukin-33 (IL-33) was recently shown to be involved in the inflammatory tumour microenvironment and the progression of colorectal cancer (CRC). We report here that the expression level of sST2, a soluble form of the IL-33 receptor (ST2L), is inversely associated with the malignant growth of CRC. sST2 is downregulated in high-metastatic cells compared with low-metastatic human and mouse CRC cells. Knockdown of sST2 in low-metastatic cells enhances tumour growth, metastasis and tumour angiogenesis, whereas its overexpression in high-metastatic cells suppresses these processes. Circulating and intratumourally administered sST2-Fc fusion protein reduce tumour growth, metastatic spread and tumour angiogenesis in mice bearing high-metastatic CRC. Mechanistically, sST2 suppresses IL-33-induced angiogenesis, Th1- and Th2-responses, macrophage infiltration and macrophage M2a polarization. In conclusion, we show that sST2 negatively regulates tumour growth and the metastatic spread of CRC through modification of the tumour microenvironment. Thus, the IL-33/ST2L axis may be a potential therapeutic target in CRC.

The antioxidant N-acetyl cysteine suppresses lidocaine-induced intracellular reactive oxygen species production and cell death in neuronal SH-SY5Y cells.

BACKGROUND: The local anesthetic lidocaine can affect intra- and extra-cellular signaling pathways in both neuronal and non-neuronal cells, resulting in long-term modulation of biological functions, including cell growth and death. Indeed, lidocaine was shown to induce necrosis and apoptosis in vitro. While several studies have suggested that lidocaine-induced apoptosis is mitochondrial pathway-dependent, it remains unclear whether reactive oxygen species (ROS) are involved in this process and whether the observed cell death can be prevented by antioxidant treatment. METHODS: The effects of lidocaine and antioxidants on cell viability and death were evaluated using SH-SY5Y cells, HeLa cells, and HeLa cell derivatives. Cell viability was examined via MTS/PES ([3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt]/phenazine ethosulfate) assay. Meanwhile, cell apoptosis and necrosis were evaluated using a cell death detection assay with Annexin V-FITC and PI staining, as well as by assaying for caspase-3/7 and caspase-9 activity, and by measuring the release of lactate dehydrogenase, respectively. Mitochondrial transmembrane potential (ΔΨm) was assessed using the fluorescent probe tetramethylrhodamine ethyl ester. RESULTS: Lidocaine treatment resulted in suppression of the mitochondrial electron transport chain and subsequent attenuation of mitochondrial membrane potential, as well as enhanced ROS production, activation of caspase-3/7 and caspase-9, and induction of apoptosis and necrosis in SH-SY5Y cells in a dose- and time-dependent manner. Likewise, the anesthetics mepivacaine and bupivacaine also induced apoptosis in SH-SY5Y cells. Notably, the antioxidants N-acetyl cysteine (NAC) and Trolox successfully scavenged the mitochondria-derived ROS and suppressed local lidocaine-induced cell death. CONCLUSIONS: Our findings demonstrate that the local anesthetics lidocaine, mepivacaine, and bupivacaine inhibited the activity of mitochondria and induced apoptosis and necrosis in a dose-dependent manner. Furthermore, they demonstrate that treatment with the antioxidants NAC, Trolox, and GGA resulted in preservation of mitochondrial voltage and inhibition of apoptosis via suppression of caspase activation.