OSU-A9 induced-reactive oxygen species cause cytotoxicity in duodenal and gastric cancer cells by decreasing phosphorylated nuclear pyruvate kinase M2 protein levels.

Pyruvate kinase M2 (PKM2) is a key enzyme responsible for the final step of glycolysis. It is still unclear whether PKM2 is involved in reactive oxygen species (ROS)-mediated cytotoxicity in gastrointestinal cancer, and what mechanisms are involved. One duodenal (AZ521) and two gastric (NUGC and SCM-1) cancer cell lines were treated with an indole-3-carbinol derivative OSU-A9, which caused cytotoxicity in acute myeloid leukemia through ROS generation. OSU-A9 caused a dose- and time-dependent cytotoxicity and induced apoptosis in duodenal and gastric cancer cells through ROS generation. Pretreatment with ROS scavengers rescued cancer cells from apoptosis and concomitant poly (ADP-ribose) polymerase cleavage, implying a key role of ROS in OSU-A9-induced cell death. Moreover, OSU-A9-induced ROS generation decreased protein levels of pTyr105-PKM2, and this effect was rescued by pretreatment with ROS scavengers. Interestingly, pTyr105-PKM2 protein levels decreased in the cell nucleus rather than in the cytoplasm. PKM2 overexpression partially rescued the survival of duodenal and gastric cancer cells treated with OSU-A9. Furthermore, the anticancer activity of OSU-A9 extended in vivo, as OSU-A9 administered by oral gavage suppressed the growth of AZ521 xenograft tumors in nude mice without obvious toxicity. In conclusion, OSU-A9 inhibited duodenal and gastric cancer cell proliferation through ROS generation and caused a subsequent decrease in nuclear pTyr105-PKM2 protein. These findings provide evidence for the non-canonical activity of PKM2 in cancer cell survival. Furthermore, they highlight the potential role of PKM2 as a future therapeutic target for duodenal and gastric cancer.

Ursolic acid induces apoptosis and autophagy in oral cancer cells.

Oral squamous cell carcinoma (OSCC) is the fifth common cause of cancer mortality in Taiwan with high incidence and recurrence and needs new therapeutic strategies. In this study, ursolic acid (UA), a triterpenoid, was examined the antitumor potency in OSCC cells. Our results showed that UA inhibited the proliferation of OSCC cells in a dose- and time-dependent manner in both Ca922 and SCC2095 oral cancer cells. UA induced caspase-dependent apoptosis accompanied with the modulation of various biological biomarkers including downregulating Akt/mTOR/NF-κB signaling, ERK, and p38. In addition, UA inhibited angiogenesis as evidenced by abrogation of migration/invasion and blocking MMP-2 secretion in Ca922 cells. Interestingly, UA induced autophagy in OSCC cells, as manifested by LC3B-II conversion and increased p62 expression and accumulation of autophagosomes. Inhibition by autophagy inhibitor enhanced UA-mediated apoptosis in Ca922 cells. The experiment provides a rationale for using triterpenoid in the treatment of OSCC.

T315 Decreases Acute Myeloid Leukemia Cell Viability through a Combination of Apoptosis Induction and Autophagic Cell Death.

T315, an integrin-linked kinase (ILK) inhibitor, has been shown to suppress the proliferation of breast cancer, stomach cancer and chronic lymphocytic leukemia cells. Here we demonstrate that T315 decreases cell viability of acute myeloid leukemia (AML) cell lines (HL-60 and THP-1) and primary leukemia cells from AML patients in a dose-responsive manner. Normal human bone marrow cells are less sensitive than leukemia cells to T315. T315 down regulates protein kinase B (Akt) and p-Akt and induces caspase activation, poly-ADP-ribose polymerase (PARP) cleavage, apoptosis and autophagy through an ILK-independent manner. Interestingly, pretreatment with autophagy inhibitors rescues cells from apoptosis and concomitant PARP cleavage, which implicates a key role of autophagic cell death in T315-mediated cytotoxicity. T315 also demonstrates efficacy in vivo, suppressing the growth of THP-1 xenograft tumors in athymic nude mice when administered intraperitoneally. This study shows that autophagic cell death and apoptosis cooperatively contribute to the anticancer activity of T315 in AML cells. In conclusion, the complementary roles of apoptotic and autophagic cell death should be considered in the future assessment of the translational value of T315 in AML therapy.

BX795, a TBK1 inhibitor, exhibits antitumor activity in human oral squamous cell carcinoma through apoptosis induction and mitotic phase arrest.

TANK-binding kinase 1 (TBK1), a member of IκB Kinase (IKK)-related kinases, plays a role in regulating innate immunity, inflammation and oncogenic signaling. This study aims to investigate the role of BX795, an inhibitor of TBK1, in a panel of oral squamous cell carcinoma (OSCC) cell lines. The antitumor effects and mechanisms of BX795 were assessed by MTT assays, flow cytometry, Western blotting, and confocal microscopy. BX795 exhibited a dose-responsive antiproliferative effect on OSCC cells with relative sparing of normal human oral keratinocytes. The compound caused apoptosis as evidenced by PARP cleavage, the presence of pyknotic nuclei in the TUNEL assay, and fragmented DNA tails in the Comet assay. BX795 inhibits Akt and NF-κB signaling, arrests cells in the mitotic phase, and increases generation of autophagy in oral cancer cells. Interestingly, the antiproliferative activity of BX795 does not correlate with TBK1 protein expression level in OSCC cells. We propose that the TBK1-independet effect is related to mitotic phase arrest. Pleiotropic anticancer activity with relative sparing of normal oral keratinocytes underscores the potential value of BX795 and warrants its further study in oral squamous cell carcinoma therapy.

Remote control of therapeutic T cells through a small molecule-gated chimeric receptor.

There is growing interest in using engineered cells as therapeutic agents. For example, synthetic chimeric antigen receptors (CARs) can redirect T cells to recognize and eliminate tumor cells expressing specific antigens. Despite promising clinical results, these engineered T cells can exhibit excessive activity that is difficult to control and can cause severe toxicity. We designed "ON-switch" CARs that enable small-molecule control over T cell therapeutic functions while still retaining antigen specificity. In these split receptors, antigen-binding and intracellular signaling components assemble only in the presence of a heterodimerizing small molecule. This titratable pharmacologic regulation could allow physicians to precisely control the timing, location, and dosage of T cell activity, thereby mitigating toxicity. This work illustrates the potential of combining cellular engineering with orthogonal chemical tools to yield safer therapeutic cells that tightly integrate cell-autonomous recognition and user control.

Synthetic biology approaches to engineer T cells.

There is rapidly growing interest in learning how to engineer immune cells, such as T lymphocytes, because of the potential of these engineered cells to be used for therapeutic applications such as the recognition and killing of cancer cells. At the same time, our knowhow and capability to logically engineer cellular behavior is growing rapidly with the development of synthetic biology. Here we describe how synthetic biology approaches are being used to rationally alter the behavior of T cells to optimize them for therapeutic functions. We also describe future developments that will be important in order to construct safe and precise T cell therapeutics.

Antitumor effects of BI-D1870 on human oral squamous cell carcinoma.

PURPOSE: Among the signaling pathways implicated in the tumorigenesis of oral squamous cell carcinoma (OSCC) is the extracellular signal-regulated kinase mitogen-activated protein kinase pathway, a downstream target of which is a family of serine/threonine kinases known as the 90 kDa ribosomal S6 kinases (RSKs). This study aims to investigate the role of BI-D1870, a specific inhibitor of p90 RSKs, in a panel of OSCC cell lines. METHODS: The antitumor effects and mechanisms of BI-D1870 were assessed by MTT assays, flow cytometry, Western blotting, transfection, and confocal microscopy. RESULTS: BI-D1870 exhibited a dose-responsive antiproliferative effect on OSCC cells with relative sparing of normal human oral keratinocytes. The compound inhibited the downstream RSK target YB-1 and caused apoptosis as evidenced by PARP cleavage, activation of the caspase cascade, and the presence of pyknotic nuclei in the 4,6-diamidino-2-phenylindole assay. In addition, BI-D1870 also induced G2/M arrest by modulating the expression of p21 and other cell cycle regulators. Other newly discovered anticancer attributes of BI-D1870 included the generation of reactive oxygen species and increases in endoplasmic reticulum stress and autophagy. CONCLUSIONS: Together, these results suggest the translational value of BI-D1870 in oral squamous cell carcinoma therapy.

OSU-A9, an indole-3-carbinol derivative, induces cytotoxicity in acute myeloid leukemia through reactive oxygen species-mediated apoptosis.

Indole-3-carbinol (I3C) is a broadly targeted phytochemical shown to prevent carcinogenesis in animal studies and to suppress the proliferation of cancer cells of human breast, colon, prostate, and endometrium. Here we demonstrate that OSU-A9, an I3C derivative with improved anticancer potency, induces cytotoxicity in acute myeloid leukemia (AML) cell lines (HL-60 and THP-1) and primary leukemia cells from AML patients in a dose-responsive manner. Normal human bone marrow cells were less sensitive to OSU-A9 than leukemia cells. OSU-A9 induces caspase activation, PARP cleavage, and autophagy but not autophagic cell death. Interestingly, pretreatment of AML cell lines and primary AML cells with N-acetylcysteine or glutathione rescues them from apoptosis (and concomitant PARP cleavage) and Akt hypophosphorylation, implicating a key role of reactive oxygen species (ROS) in OSU-A9-related cytotoxicity. Importantly, the anticancer utility of OSU-A9 is extended in vivo as it, administered intraperitoneally, suppresses the growth of THP-1 xenograft tumors in athymic nude mice without obvious toxicity. This study shows that ROS-mediated apoptosis contributes to the anticancer activity of OSU-A9 in AML cell lines and primary AML cells, and thus should be considered in the future assessment of its translational value in AML therapy.

Cucurbitane Triterpenoid from Momordica charantia Induces Apoptosis and Autophagy in Breast Cancer Cells, in Part, through Peroxisome Proliferator-Activated Receptor γ Activation.

Although the antitumor activity of the crude extract of wild bitter gourd (Momordica charantia L.) has been reported, its bioactive constituents and the underlying mechanism remain undefined. Here, we report that 3 ß ,7 ß -dihydroxy-25-methoxycucurbita-5,23-diene-19-al (DMC), a cucurbitane-type triterpene isolated from wild bitter gourd, induced apoptotic death in breast cancer cells through peroxisome proliferator-activated receptor (PPAR) γ activation. Luciferase reporter assays indicated the ability of DMC to activate PPAR γ , and pharmacological inhibition of PPAR γ protected cells from DMC's antiproliferative effect. Western blot analysis indicated that DMC suppressed the expression of many PPAR γ -targeted signaling effectors, including cyclin D1, CDK6, Bcl-2, XIAP, cyclooxygenase-2, NF- κ B, and estrogen receptor α , and induced endoplasmic reticulum stress, as manifested by the induction of GADD153 and GRP78 expression. Moreover, DMC inhibited mTOR-p70S6K signaling through Akt downregulation and AMPK activation. The ability of DMC to activate AMPK in liver kinase (LK) B1-deficient MDA-MB-231 cells suggests that this activation was independent of LKB1-regulated cellular metabolic status. However, DMC induced a cytoprotective autophagy presumably through mTOR inhibition, which could be overcome by the cotreatment with the autophagy inhibitor chloroquine. Together, the ability of DMC to modulate multiple PPAR γ -targeted signaling pathways provides a mechanistic basis to account for the antitumor activity of wild bitter gourd.

Inhibition of hedgehog signaling induces monocytic differentiation of HL-60 cells.

There is little evidence to demonstrate the importance of the Sonic hedgehog homolog (Shh) pathway to differentiation therapy in the treatment of hematological neoplasms. Here we characterize the changes in acute myelogenous leukemia (HL-60) cells after blocking the Shh pathway by an antagonist of Smoothened, cyclopamine. Cyclopamine induces apoptosis of HL-60 cells in a dose- and time-dependent manner with increased G0/G1 cycle fraction. Treatment with cyclopamine increases the expression of monocytic cell markers CD11b and CD14, but the expression of CD13, CD33 and CD38 is unchanged. The monocytic differentiation of HL-60 cells induced by cyclopamine is also evidenced by an increase in Egr-1 expression. Importantly, cyclopamine down-regulates the phosphorylation of Akt and ERK, but activates AMP-activated protein kinase (AMPK) signaling. Further investigations should determine the clinical application of modulating the Shh pathway in the treatment of hematological malignancies.

Dietary zerumbone prevents mouse cornea from UVB-induced photokeratitis through inhibition of NF-κB, iNOS, and TNF-α expression and reduction of MDA accumulation.

PURPOSE: Ultraviolet B (UVB) irradiation activates nuclear factor-kappa B (NF-κB) and inducible nitric oxide synthase (iNOS) in the cornea, resulting in inflammatory responses and malondialdehyde (MDA) accumulation. This study aims to determine the effect of zerumbone, a potent NF-κB inhibitor and inflammation modulators, on UVB-induced corneal damages in a mouse model. METHODS: Fifty female imprinting control region (ICR) mice were randomly divided into five groups. The mice were anaesthetized with their ocular surfaces exposed to UVB light (0.72J/cm(2)/daily), followed by daily dietary zerumbone supplements at 0, 1, 10, and 100 mg/kg of bodyweight. Mice without zerumbone supplements were used as treatment controls and mice without UVB irradiation as blank controls. Corneal surface damages were graded according to smoothness, opacity, and the extent of lissamine green staining. Histopathological changes were also examined, along with the expression of NF-κB, iNOS, and tumor necrosis factor-α (TNF-α). MDA accumulation and the levels of two antioxidant enzymes, glutathione (GSH) and GSH reductase (GR) were also examined. RESULTS: UVB irradiation caused significant damages to cornea, including sustained inflammation, apparent corneal ulcer, and severe epithelial exfoliation, leading to thinning of corneal epithelial layer, and infiltration of polymorphonuclear leukocytes. NF-κB expression was highly activated with nuclear translocation. The expression of iNOS and TNF-α were increased. MDA accumulation was also increased in both the corneal epithelial layer and the stroma. With dietary zerumbone, corneal damages were ameliorated in a dose-dependent manner. NF-κB activation and its nuclear translocation were blocked with decreased expression of iNOS and TNF-α. Infiltration of polymorphonuclear leukocytes was also blocked by dietary zerumbone. Besides, MDA accumulation was reduced with concomitant increase of GSH and GR levels. CONCLUSIONS: Dietary zerumbone prevents UVB-induced corneal damages by inhibition of NF-κB, iNOS, and TNF-α, with concomitant reduction of MDA accumulation and increase of GSH and GR levels in the mouse model. Results of this study suggest that dietary zerumbone may be used as a prophylactic agent against UVB-induced photokeratitis.

Dietary zerumbone prevents against ultraviolet B-induced cataractogenesis in the mouse.

PURPOSE: To investigate the preventive effect of dietary zerumbone against UVB-induced cataractogenesis. METHODS: A total of 50 six-week-old female ICR mice were split into five groups (each contained 10 mice) and exposed to UVB (0.72 J/cm(2)/daily) at noon for 7 days, except for the blank control group. The mice with UVB exposure were fed with zerumbone as a dietary supplement at 0, 1, 10, and 100 mg/kg of bodyweight, respectively, starting from one day before UVB exposure. On day 7, at 4 h after UVB exposure, all mice were subjected to cataract examination and lens opacity scoring, in correlation with levels of MDA (malondialdehyde), GSH (glutathione), GR (GSH reductase), GPx (glutathione peroxidase), and SOD (superoxide dismutase) in the lens. RESULTS: Dietary zerumbone at 100 mg/kg after UVB exposure was effective in decreasing lens opacity scores (p<0.001) and to reduce MDA (p<0.001), while GSH and GR levels were significantly increased (both p<0.001) in the lens. SOD was also increased with dietary zerumbone at 100 mg/kg (p=0.115), whereas GPx (p=0.171) levels were lower as compared with those without zerumbone after UVB exposure. CONCLUSIONS: These results suggest that zerumbone may protect against UVB-induced cataractogensis through reducing lipid peroxides and enhancing the endogenous antioxidant GSH level and GR activity.

Hedgehog overexpression leads to the formation of prostate cancer stem cells with metastatic property irrespective of androgen receptor expression in the mouse model.

BACKGROUND: Hedgehog signalling has been implicated in prostate tumorigenesis in human subjects and mouse models, but its effects on transforming normal basal/stem cells toward malignant cancer stem cells remain poorly understood. METHODS: We produced pCX-shh-IG mice that overexpress Hedgehog protein persistently in adult prostates, allowing for elucidation of the mechanism during prostate cancer initiation and progression. Various markers were used to characterize and confirm the transformation of normal prostate basal/stem cells into malignant cancer stem cells under the influence of Hedgehog overexpression. RESULTS: The pCX-shh-IG mice developed prostatic intraepithelial neoplasia (PIN) that led to invasive and metastatic prostate cancers within 90 days. The prostate cancer was initiated through activation of P63+ basal/stem cells along with simultaneous activation of Hedgehog signalling members, suggesting that P63+/Patch1+ and P63+/Smo+ cells may serve as cancer-initiating cells and progress into malignant prostate cancer stem cells (PCSCs). In the hyperplastic lesions and tumors, the progeny of PCSCs differentiated into cells of basal-intermediate and intermediate-luminal characteristics, whereas rare ChgA+ neuroendocrine differentiation was seen. Furthermore, in the metastatic loci within lymph nodes, kidneys, and lungs, the P63+ PCSCs formed prostate-like glandular structures, characteristic of the primitive structures during early prostate development. Besides, androgen receptor (AR) expression was detected heterogeneously during tumor progression. The existence of P63+/AR-, CK14+/AR- and CD44+/AR- progeny indicates direct procurement of AR- malignant cancer trait. CONCLUSIONS: These data support a cancer stem cell scenario in which Hedgehog signalling plays important roles in transforming normal prostate basal/stem cells into PCSCs and in the progression of PCSCs into metastatic tumor cells.

Control of transcription by cell size.

Cell size increases significantly with increasing ploidy. Differences in cell size and ploidy are associated with alterations in gene expression, although no direct connection has been made between cell size and transcription. Here we show that ploidy-associated changes in gene expression reflect transcriptional adjustment to a larger cell size, implicating cellular geometry as a key parameter in gene regulation. Using RNA-seq, we identified genes whose expression was altered in a tetraploid as compared with the isogenic haploid. A significant fraction of these genes encode cell surface proteins, suggesting an effect of the enlarged cell size on the differential regulation of these genes. To test this hypothesis, we examined expression of these genes in haploid mutants that also produce enlarged size. Surprisingly, many genes differentially regulated in the tetraploid are identically regulated in the enlarged haploids, and the magnitude of change in gene expression correlates with the degree of size enlargement. These results indicate a causal relationship between cell size and transcription, with a size-sensing mechanism that alters transcription in response to size. The genes responding to cell size are enriched for those regulated by two mitogen-activated protein kinase pathways, and components in those pathways were found to mediate size-dependent gene regulation. Transcriptional adjustment to enlarged cell size could underlie other cellular changes associated with polyploidy. The causal relationship between cell size and transcription suggests that cell size homeostasis serves a regulatory role in transcriptome maintenance.

Congenital eye malformations associated with extensive periocular neural crest apoptosis after influenza B virus infection during early embryogenesis.

PURPOSE: Congenital eye malformations are a leading cause of blindness in children. Influenza virus infections prevail worldwide and have been implicated in congenital defects. Infections acquired during gestation may disrupt eye morphogenesis. We investigated the effects of influenza B virus infection on eye malformations during early embryogenesis. METHODS: Chick embryos were exposed to influenza B virus at Hamburger-Hamilton stage 9. Maternal infection was conducted by exposing pregnant ICR mice to influenza B virus at the embryonic gestation stage E 5.0. After infection, virus RNA distribution was detected by in situ hybridization at various developmental stages. The distribution of periocular neural crest cells and the extent of apoptosis were examined by immunohistochemical staining, in correlation with eye malformations. RESULTS: Microphthalmos and anophthalmos, together with neural tube defects, were found in the chick and mouse embryos following the infections. The viral RNA was detected in the head neuroepithelium, optic vesicle, periocular mesenchyme, and the forming ventricles of the developing brain. Immunohistochemical staining revealed aberrant neural crest distribution and extensive apoptosis in the head surface ectoderm, periocular mesenchyme, and optic vesicle in the chick embryos. Furthermore, transplacental infection was confirmed by the presence of viral RNA in the mouse fetuses, with eye and neural tube defects similar to those found in the chick embryos after experimental infections. CONCLUSIONS: Influenza B virus may act as a teratogen to cause aberrant periocular neural crest cell contribution to eye development and extensive apoptosis, resulting in congenital eye malformations.

Mlp-dependent anchorage and stabilization of a desumoylating enzyme is required to prevent clonal lethality.

Myosin-like proteins 1 and 2 (Mlp1 and Mlp2) form filaments attached to the nucleoplasmic side of the nuclear pore complexes via interaction with the nucleoporin Nup60. Here, we show that Mlps and Nup60, but not several other nucleoporins, are required to localize and stabilize a desumoylating enzyme Ulp1. Moreover, like Mlps, Ulp1 exhibits a unique asymmetric distribution on the nuclear envelope. Consistent with a role in regulating Ulp1, removal of either or both MLPs affects the SUMO conjugate pattern. We also show that deleting MLPs or the localization domains of Ulp1 results in DNA damage sensitivity and clonal lethality, the latter of which is caused by increased levels of 2-micron circle DNA. Epistatic and dosage suppression analyses further demonstrate that Mlps function upstream of Ulp1 in 2-micron circle maintenance and the damage response. Together, our results reveal that Mlps play important roles in regulating Ulp1 and subsequently affect sumoylation stasis, growth, and DNA repair.