Synthesis and biological evaluation of echinomycin analogues as potential colon cancer agent.

Colorectal cancer is the third most commonly diagnosed cancer and the second leading cause of cancer-related death, thus a novel chemotherapeutic agent for colon cancer therapy is needed. In this study, analogues of echinomycin, a cyclic peptide natural product with potent toxicity to several human cancer cell lines, were synthesized, and their biological activities against human colon cancer cells were investigated. Analogue 3 as well as 1 inhibit HIF-1α-mediated transcription. Notably, transcriptome analysis indicated that the cell cycle and its regulation were involved in the effects on cells treated with 3. Analogue 3 exhibited superior in vivo efficacy to echinomycin without significant toxicity in mouse xenograft model. The low dose of 3 needed to be efficacious in vivo is also noteworthy and our data suggest that 3 is an attractive and potentially novel agent for the treatment of colon cancer.

Structural and functional analyses of the echinomycin resistance conferring protein Ecm16 from Streptomyces lasalocidi.

Echinomycin is a natural product DNA bisintercalator antibiotic. The echinomycin biosynthetic gene cluster in Streptomyces lasalocidi includes a gene encoding the self-resistance protein Ecm16. Here, we present the 2.0 Å resolution crystal structure of Ecm16 bound to adenosine diphosphate. The structure of Ecm16 closely resembles that of UvrA, the DNA damage sensor component of the prokaryotic nucleotide excision repair system, but Ecm16 lacks the UvrB-binding domain and its associated zinc-binding module found in UvrA. Mutagenesis study revealed that the insertion domain of Ecm16 is required for DNA binding. Furthermore, the specific amino acid sequence of the insertion domain allows Ecm16 to distinguish echinomycin-bound DNA from normal DNA and link substrate binding to ATP hydrolysis activity. Expression of ecm16 in the heterologous host Brevibacillus choshinensis conferred resistance against echinomycin and other quinomycin antibiotics, including thiocoraline, quinaldopeptin, and sandramycin. Our study provides new insight into how the producers of DNA bisintercalator antibiotics fend off the toxic compounds that they produce.

Prolonged stretching of rat uteri causes hypoxia and inhibits contractility via potassium channel TREK1.

In brief: During pregnancy, uterine kept quiescence along with uterine overdistention before labor. Prolonged stretching induced uterus myometrial hypoxia, increased TREK1 expression, and relaxed the myometrium, which may contribute to uterine quiescence and atony during pregnancy. Abstract: The mechanisms underlying pre-labor uterine quiescence and uterine atony during overdistention are unclear. TREK1 (a two-pore domain potassium channel) and hypoxia-inducible factor-1α (HIF-1α) are activated by mechanical stretch, and their expression is upregulated by decreased uterine contractility. HIF-1α is a nuclear factor which regulates numerous target proteins, but whether it regulates TREK1 during the uterine stretch to cause uterine quiescence and/or atony is unclear. We investigated uterine contractility at different gestational stages in rats, as well as in non-pregnant uteri, which were induced by prolonged stretching and hypoxia. We also assessed the effects of incubating the uteri with or without echinomycin or l-methionine. Moreover, we analyzed HIF-1α and TREK1 expression levels in each group, as well as at various gestational stages of pregnant human uteri. We found that contractility was significantly decreased in pregnant uteri when compared with non-pregnant uteri, and this decrease was associated with increases in HIF-1α and TREK1 expression levels. HIF-1α and TREK1 expression levels in human uteri increased with the gestational length. Decreased uterine contractility and increased HIF-1α and TREK1 expression levels were also observed in non-pregnant rat uteri under 8 g of stretching tension or hypoxia. Inhibition of hypoxia with echinomycin restored normal uterine contractility, while HIF-1α and TREK1 protein expression remained reduced. TREK1 inhibition with l-methionine also restored uterine contractility under tension or hypoxia. In conclusion, we demonstrated that prolonged stretching induces myometrial hypoxia, increases TREK1 expression, and relaxes the myometrium, which may contribute to uterine quiescence and atony.

Echinomycin as a promising therapeutic agent against KSHV-related malignancies.

Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiologic agent of several human cancers, including Kaposi's sarcoma (KS) and primary effusion lymphoma (PEL), which preferentially arise in immunocompromised patients while lack of effective therapeutic options. Oncoproteins Myc and hypoxia-inducible factor-1α (HIF1α) have been found closely related to KSHV infection, replication and oncogenesis. However, the strategies of dual targeting these two oncoproteins have never been developed and tested for treatments of KSHV-related malignancies. In the current study, we report that treatment of echinomycin dramatically regresses cell growth both in vitro-cultured KSHV + tumor cells and in vivo KS or PEL xenograft mice models, through simultaneously inhibiting Myc and HIF1α expression. Echinomycin treatment also induces viral lytic gene expression whereas not increasing infectious virions production from KSHV + tumor cells. Our comparative transcriptomic analysis has identified a bunch of new Echinomycin-regulated, Myc- and HIF1α-related genes contributed to KSHV pathogenesis, including KDM4B and Tau, which are required for the survival of KSHV + tumor cells with functional validation. These data together reveal that dual targeting Myc and HIF1α such as using Echinomycin may represent a new and promising option for treatments of these virus-associated malignancies.

The UvrA-like protein Ecm16 requires ATPase activity to render resistance against echinomycin.

Bacteria use various strategies to become antibiotic resistant. The molecular details of these strategies are not fully understood. We can increase our understanding by investigating the same strategies found in antibiotic-producing bacteria. In this work, we characterize the self-resistance protein Ecm16 encoded by echinomycin-producing bacteria. Ecm16 is a structural homolog of the nucleotide excision repair protein UvrA. Expression of ecm16 in the heterologous system Escherichia coli was sufficient to render resistance against echinomycin. Ecm16 binds DNA (double-stranded and single-stranded) using a nucleotide-independent binding mode. Ecm16's binding affinity for DNA increased by 1.7-fold when the DNA is intercalated with echinomycin. Ecm16 can render resistance against echinomycin toxicity independently of the nucleotide excision repair system. Similar to UvrA, Ecm16 has ATPase activity, and this activity is essential for Ecm16's ability to render echinomycin resistance. Notably, UvrA and Ecm16 were unable to complement each other's function. Together, our findings identify new mechanistic details of how a refurbished DNA repair protein Ecm16 can specifically render resistance to the DNA intercalator echinomycin.

Antitumor effect of the selective hypoxia-inducible factor-1 inhibitors echinomycin and PX-478 on uterine fibroids.

OBJECTIVE: To investigate the antitumor effects of the selective hypoxia-inducible factor-1 (HIF-1) inhibitors echinomycin and PX-478 on uterine fibroids. DESIGN: Experimental study using in vitro primary culture systems and an in vivo mouse xenograft model. SETTING: Academic university center. PATIENT(S): Women with uterine fibroids who underwent hysterectomy or myomectomy. INTERVENTION(S): Administration of the selective HIF-1 inhibitors echinomycin and PX-478 to the media of the primary cultured uterine fibroid cells and to nonobese diabetic/severe combined immunodeficient mice bearing fibroid xenografts consisting of the primary cultured fibroid cells and type Ⅰ collagen gels beneath the kidney capsule. MAIN OUTCOME MEASURE(S): Cell proliferation was measured by Cell Counting Kit-8 assay. Apoptosis was evaluated by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay and by measuring caspase 3 and 7 activities. The xenografts were evaluated by gross appearance, surface area, and histology. The Ki-67 index was measured to evaluate proliferation of the xenografts. RESULT(S): Both echinomycin and PX-478 inhibited cell proliferation and induced apoptosis in fibroid cells cultured under hypoxia and normoxia. Enlargement of the fibroid xenografts was significantly attenuated. The Ki-67 index significantly decreased after the administration of the HIF-1 inhibitors in the xenograft model. Eight of 27 xenografts treated with the HIF-1 inhibitors contained calcification and hyalinizing components from 3 days after the grafting to 2 weeks, suggesting that the HIF-inhibitors induce degeneration of the fibroid xenografts. CONCLUSION(S): The selective HIF-1 inhibitors echinomycin and PX-478 show antitumor effects against uterine fibroids both in vitro and in vivo. These findings support the potential use of HIF-1 inhibitors for the treatment of uterine fibroids.

Targeting HIF-1α abrogates PD-L1-mediated immune evasion in tumor microenvironment but promotes tolerance in normal tissues.

A combination of anti-CTLA-4 plus anti-PD-1/PD-L1 is the most effective cancer immunotherapy but causes high incidence of immune-related adverse events (irAEs). Here we report that targeting of HIF-1α suppressed PD-L1 expression on tumor cells and tumor-infiltrating myeloid cells, but unexpectedly induced PD-L1 in normal tissues by an IFN-γ-dependent mechanism. Targeting the HIF-1α/PD-L1 axis in tumor cells reactivated tumor-infiltrating lymphocytes and caused tumor rejection. The HIF-1α inhibitor echinomycin potentiated the cancer immunotherapeutic effects of anti-CTLA-4 therapy, with efficacy comparable to that of anti-CTLA-4 plus anti-PD-1 antibodies. However, while anti-PD-1 exacerbated irAEs triggered by ipilimumab, echinomycin protected mice against irAEs by increasing PD-L1 levels in normal tissues. Our data suggest that targeting HIF-1α fortifies the immune tolerance function of the PD-1/PD-L1 checkpoint in normal tissues but abrogates its immune evasion function in the tumor microenvironment to achieve safer and more effective immunotherapy.

Pharmacological or genetic inhibition of hypoxia signaling attenuates oncogenic RAS-induced cancer phenotypes.

Oncogenic Ras mutations are highly prevalent in hematopoietic malignancies. However, it is difficult to directly target oncogenic RAS proteins for therapeutic intervention. We have developed a Drosophila acute myeloid leukemia model induced by human KRASG12V, which exhibits a dramatic increase in myeloid-like leukemia cells. We performed both genetic and drug screens using this model. The genetic screen identified 24 candidate genes able to attenuate the oncogenic RAS-induced phenotype, including two key hypoxia pathway genes HIF1A and ARNT (HIF1B). The drug screen revealed that echinomycin, an inhibitor of HIF1A, can effectively attenuate the leukemia phenotype caused by KRASG12V. Furthermore, we showed that echinomycin treatment can effectively suppress oncogenic RAS-driven leukemia cell proliferation, using both human leukemia cell lines and a mouse xenograft model. These data suggest that inhibiting the hypoxia pathway could be an effective treatment approach and that echinomycin is a promising targeted drug to attenuate oncogenic RAS-induced cancer phenotypes. This article has an associated First Person interview with the first author of the paper.

Characterization of Streptomyces sp. LS462 with high productivity of echinomycin, a potent antituberculosis and synergistic antifungal antibiotic.

A biologically active microbial strain, designated as "LS462," was isolated from a soil sample collected from Yaoli Virgin Forest of Jiangxi Province, China. The strain was able to produce a high yield of echinomycin (172 mg/l) even under nonoptimized culture conditions and is proposed to serve as a promising source of echinomycin. In this study, echinomycin exhibited strong anti-Mycobacterium tuberculosis H37Rv activity and synergistic antifungal effect with a greatly reduced dosage of posaconazole on Candida albicans SC5314. The strain belongs to the genus Streptomyces according to its morphological and 16S rDNA phylogenetic analysis. The 16S rDNA was found to have the highest sequence identity with Streptomyces fuscichromogenes (99.37% similarity). Extensive nuclear magnetic resonance and mass spectroscopic data were used to determine the structure of echinomycin. The strain S. fuscichromogenes has not been previously reported to produce echinomycin. Strain LS462 may be exploited as a new potential source for the commercial production of echinomycin. Also, this work is the first to report the new synergistic antifungal activity of echinomycin and further study of the synergistic mechanism will be helpful to guide the development of antifungal agents.

Quinomycin A reduces cyst progression in polycystic kidney disease.

Polycystic kidney disease (PKD) is a genetic disorder characterized by aberrant renal epithelial cell proliferation and formation and progressive growth of numerous fluid-filled cysts within the kidneys. Previously, we showed that there is elevated Notch signaling compared to normal renal epithelial cells and that Notch signaling contributes to the proliferation of cystic cells. Quinomycin A, a bis-intercalator peptide, has previously been shown to target the Notch signaling pathway and inhibit tumor growth in cancer. Here, we show that Quinomycin A decreased cell proliferation and cyst growth of human ADPKD cyst epithelial cells cultured within a 3D collagen gel. Treatment with Quinomycin A reduced kidney weight to body weight ratio and decreased renal cystic area and fibrosis in Pkd1RC/RC ; Pkd2+/- mice, an orthologous PKD mouse model. This was accompanied by reduced expression of Notch pathway proteins, RBPjk and HeyL and cell proliferation in kidneys of PKD mice. Quinomycin A treatments also normalized cilia length of cyst epithelial cells derived from the collecting ducts. This is the first study to demonstrate that Quinomycin A effectively inhibits PKD progression and suggests that Quinomycin A has potential therapeutic value for PKD patients.

Echinomycin mitigates ocular angiogenesis by transcriptional inhibition of the hypoxia-inducible factor-1.

BACKGROUND: Echinomycin (EKN), an inhibitor of hypoxia-inducible factor (HIF)-1 DNA-binding activity, has been implied as a possible therapeutic agent in ischemic diseases. Here, we assess EKN in hypoxia-driven responses in vitro using human primary adult retinal pigment epithelium cells (aRPE) and retinal endothelial cells (hREC), and in vivo using the laser-induced mouse choroidal neovascularization (CNV) model. METHODS: Effects of EKN on hypoxia-mediated pathways in aRPE were analyzed by Western blotting for HIF-1α protein, quantitative PCR of HIF-target genes, and proteome array for soluble angiogenic factors. In vitro inhibition of angiogenesis by EKN was determined in hREC. In vivo inhibition of angiogenesis by EKN was determined in the mouse laser-induced CNV, as a model of HIF-associated ocular neovascularization. CNV lesion area was determined by fundus fluorescein angiography. RESULTS: aRPE treated with EKN showed hypoxia-dependent significantly decreased cell recovery in the wound healing assay. These results were supported by lower levels of HIF-mediated transcripts detected in hypoxic aRPE cells treated with EKN compared with non-treated controls, and confirmed by proteome profiler for angiogenic factors. hREC exposed to aRPE EKN-conditioned medium displayed reduced sprouting angiogenesis. Mice with laser-induced CNV treated with intravitreally injected EKN showed significantly decreased vascular lesion area when compared with a mouse equivalent of aflibercept, or vehicle-treated controls. CONCLUSIONS: Our data proposes EKN as a potent inhibitor of HIF-mediated angiogenesis in retinal cells and in the mouse model of CNV, which could have future implications in the treatment of patients with neovascular age-related macular degeneration.

Hypoxia-inducible factor (HIF1alpha) inhibition modulates cumulus cell function and affects bovine oocyte maturation in vitro†.

Various metabolic and hormonal factors expressed in cumulus cells are positively correlated with the in vitro maturation (IVM) of oocytes. However, the role of hypoxia sensing both during maturation of cumulus-oocyte complexes (COCs) as well as during the resumption of meiosis remains uncertain. HIF1alpha plays major roles in cellular responses to hypoxia, and here we investigated its role during bovine COC maturation by assessing the expression of related genes in cumulus cells. COCs were divided into the following groups: immature (control), in vitro matured (IVM/control), or matured in the presence of a blocker of HIF1alpha activity (echinomycin, IVM/E). We found an inhibition of cumulus cell expansion in IVM/E, compared with the IVM/control. Transcript levels of several factors (n = 13) were assessed in cumulus cells. Decreased expression of HAS2, TNFAIP6, TMSB4, TMSB10, GATM, GLUT1, CX43, COX2, PTGES, and STAR was found in IVM/E (P < 0.05). Additionally, decreased protein levels were detected for STAR, HAS2, and PCNA (P < 0.05), while activated-Caspase 3 remained unaffected in IVM/E. Progesterone output decreased in IVM/E. The application of PX-478, another blocker of HIF1alpha expression, yielded identical results. Negative effects of HIF1alpha suppression were further observed in the significantly decreased oocyte maturation and blastocyst rates from COCs matured with echinomycin (P < 0.05) or PX-478 (P < 0.05). These results support the importance of HIF1alpha for COC maturation and subsequent embryo development. HIF1alpha is a multidirectional factor controlling intercellular communication within COCs, steroidogenic activity, and oocyte development rates, and exerting effects on blastocyst rates.

Liposomal formulation of HIF-1α inhibitor echinomycin eliminates established metastases of triple-negative breast cancer.

Hypoxia-inducible factor 1α (HIF-1α) is recognized as a prime molecular target for metastatic cancer. However, no specific HIF-1α inhibitor has been approved for clinical use. Here, we demonstrated that in vivo efficacy of echinomycin in solid tumors with HIF-1α overexpression is formulation-dependent. Compared to previously-used Cremophor-formulated echinomycin, which was toxic and ineffective in clinical trials, liposomal-echinomycin provides significantly more inhibition of primary tumor growth and only liposome-formulated echinomycin can eliminate established triple-negative breast cancer (TNBC) metastases, which are the leading cause of death from breast cancer, as available therapies remain minimally effective at this stage. Pharmacodynamic analyses reveal liposomal-echinomycin more potently inhibits HIF-1α transcriptional activity in primary and metastasized TNBC cells in vivo, the latter of which are HIF-1α enriched. The data suggest that nanoliposomal-echinomycin can provide safe and effective therapeutic HIF-1α inhibition and could represent the most potent HIF-1α inhibitor in prospective trials for metastatic cancer.

HIF1 driven transcriptional activity regulates steroidogenesis and proliferation of bovine granulosa cells.

Hypoxia-inducible factor 1 (HIF1) is a heterodimeric transcription factor, consisting of a constitutively expressed ß-subunit (HIF1B) and a regulated α-subunit (HIF1A). In the present study, we analyzed the HIF1 driven transcriptional activity in bovine granulosa cells (GC). Treatment of GC with FSH (follicle stimulating hormone) and IGF1 (insulin-like growth factor 1) resulted in the upregulation of HIF1A mRNA expression under normoxia. Immunohistochemistry of bovine ovarian sections showed distinct staining of HIF1A in the GC layer of different staged ovarian follicles. Suppression of HIF1 using echinomycin and gene knockdown procedures revealed that HIF1 transcriptionally regulates the genes associated with steroidogenesis (STAR, HSD3B and CYP19A1) and proliferation (CCND2 and PCNA) of GC. Further, our data suggest that CYP19A1, the key gene of estradiol production, is one of the plausible downstream targets of HIF1 in bovine GC as shown by gene expression, radioimmunoassay, and chromatin precipitation analysis. Based on these results, we propose that HIF1 driven transcriptional activity plays a crucial role in GC functionality, especially steroidogenesis and proliferation in developing bovine ovarian follicles.

Therapeutic targeting of TP53-mutated acute myeloid leukemia by inhibiting HIF-1α with echinomycin.

TP53 mutation in acute myeloid leukemia (AML) is associated with poor prognosis. Since no targeted therapy is available to restore p53 function, it is of great interest to test whether other pathways activated by TP53 mutations can be therapeutically targeted. Here, we showed HIF-1α target genes are enriched in TP53-mutated versus TP53-wild-type AML. To determine the role of this activation, we tested efficacy of HIF-1α inhibitor echinomycin in TP53-mutated AML samples in vitro and in vivo. Echinomycin was broadly effective against a panel of primary AML blast cells, with low nanomolar IC50s and, based on colony-forming unit assay, was tenfold more effective in eliminating AML stem cells. Echinomycin selectively eliminated CD34+CD38- AML cells. To test the therapeutic efficacy of echinomycin, we established a xenograft model of TP53-mutated AML. Echinomycin was broadly effective against xenografts from multiple AML samples in vivo, and more effective than cytarabine + daunorubicin chemotherapy. Importantly, while cytarabine + daunorubicin enriched for AML stem cells, echinomycin nearly eliminated this population. Using TP53-mutated AML cell line THP1 and patient-derived AML cells, we tested a new echinomycin formulation with longer half-life and significantly improved therapeutic effect. Our data suggest a novel approach to treat AML with TP53 mutations.

Hypoxia-inducible factor 1 (HIF-1) is a new therapeutic target in JAK2V617F-positive myeloproliferative neoplasms.

Classical Philadelphia chromosome-negative myeloproliferative neoplasms (MPN) are a heterogeneous group of hematopoietic malignancies including polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). The JAK2V617F mutation plays a central role in these disorders and can be found in 90% of PV and ~50-60% of ET and PMF. Hypoxia-inducible factor 1 (HIF-1) is a master transcriptional regulator of the response to decreased oxygen levels. We demonstrate the impact of pharmacological inhibition and shRNA-mediated knockdown (KD) of HIF-1α in JAK2V617F-positive cells. Inhibition of HIF-1 binding to hypoxia response elements (HREs) with echinomycin, verified by ChIP, impaired growth and survival by inducing apoptosis and cell cycle arrest in Jak2V617F-positive 32D cells, but not Jak2WT controls. Echinomycin selectively abrogated clonogenic growth of JAK2V617F cells and decreased growth, survival, and colony formation of bone marrow and peripheral blood mononuclear cells and iPS cell-derived progenitor cells from JAK2V617F-positive patients, while cells from healthy donors were unaffected. We identified HIF-1 target genes involved in the Warburg effect as a possible underlying mechanism, with increased expression of Pdk1, Glut1, and others. That was underlined by transcriptome analysis of primary patient samples. Collectively, our data show that HIF-1 is a new potential therapeutic target in JAK2V617F-positive MPN.

Bortezomib and metformin opposingly regulate the expression of hypoxia-inducible factor alpha and the consequent development of chemotherapy-induced painful peripheral neuropathy.

Chemotherapy-induced painful peripheral neuropathy is a significant clinical problem that is associated with widely used chemotherapeutics. Unfortunately, the molecular mechanisms by which chemotherapy-induced painful peripheral neuropathy develops have remained elusive. The proteasome inhibitor, bortezomib, has been shown to induce aerobic glycolysis in sensory neurons. This altered metabolic phenotype leads to the extrusion of metabolites which sensitize primary afferents and cause pain. Hypoxia-inducible factor alpha is a transcription factor that is known to reprogram cellular metabolism. Furthermore, hypoxia-inducible factor 1 alpha protein is constantly synthesized and undergoes proteasomal degradation in normal conditions. However, metabolic stress or hypoxia stabilizes the expression of hypoxia-inducible factor 1 alpha leading to the transcription of genes that reprogram cellular metabolism. This study demonstrates that treatment of mice with bortezomib stabilizes the expression of hypoxia-inducible factor 1 alpha. Moreover, knockdown of hypoxia-inducible factor 1 alpha, inhibition of hypoxia-inducible factor 1 alpha binding to its response element, or limiting its translation by using metformin prevent the development of bortezomib-induced neuropathic pain. Strikingly, the blockade of hypoxia-inducible factor 1 alpha expression does not attenuate mechanical allodynia in mice with existing bortezomib-induced neuropathic pain. These results establish the stabilization of hypoxia-inducible factor 1 alpha expression as the molecular mechanism by which bortezomib initiates chemotherapy-induced painful peripheral neuropathy. Crucially, these findings reveal that the initiation and maintenance of bortezomib-induced neuropathic pain are regulated by distinct mechanisms.

Early redox activities modulate Xenopus tail regeneration.

Redox state sustained by reactive oxygen species (ROS) is crucial for regeneration; however, the interplay between oxygen (O2), ROS and hypoxia-inducible factors (HIF) remains elusive. Here we observe, using an optic-based probe (optrode), an elevated and steady O2 influx immediately upon amputation. The spatiotemporal O2 influx profile correlates with the regeneration of Xenopus laevis tadpole tails. Inhibition of ROS production but not ROS scavenging decreases O2 influx. Inhibition of HIF-1α impairs regeneration and stabilization of HIF-1α induces regeneration in the refractory period. In the regeneration bud, hypoxia correlates with O2 influx, ROS production, and HIF-1α stabilization that modulate regeneration. Further analyses reveal that heat shock protein 90 is a putative downstream target of HIF-1α while electric current reversal is a de facto downstream target of HIF-1α. Collectively, the results show a mechanism for regeneration via the orchestration of O2 influx, ROS production, and HIF-1α stabilization.

Core circadian clock gene expression in human dental pulp-derived cells in response to L-mimosine, hypoxia and echinomycin.

Core circadian clock genes set the pace for a wide range of physiological functions, including regeneration. The role of these genes and their regulation in the dental pulp, in particular under hypoxic conditions, is unknown. Here we investigated if core clock genes are expressed in human dental pulp-derived cells (DPC) and if their expression is modulated by the hypoxia mimetic agent, L-mimosine (L-MIM), hypoxia or echinomycin. Dental pulp-derived cells in monolayers and spheroids were treated with L-MIM, hypoxia or echinomycin. mRNA levels of the core circadian clock genes were analysed using quantitative PCR (qPCR) and their protein levels were analysed by western blot. All core clock genes and proteins were produced in DPC monolayer and spheroid cultures. The expression of cryptochrome circadian regulators and period circadian regulators was reduced by L-MIM, hypoxia and echinomycin at mRNA, but not at protein levels. Time course experiments indicated that modulations were based on alterations in overall mRNA levels of core circadian clock genes. Our results suggest a potential role of the core circadian clock in the response of dental pulp to hypoxia. Future studies need to consider that regulation of the core circadian clock at mRNA levels might not be paralleled by modulation of protein levels.

Quinomycins H1 and H2, new cytotoxic antibiotics from Streptomyces sp. RAL404.

Two new cytotoxic antibiotics designated quinomycins H1 (2) and H2 (3) were isolated from the culture broth of Streptomyces sp. RAL404. The molecular formula of both compounds was established as C52H65N11O13S2 by electrospray ionization mass spectrometry (ESI-MS). Their structures were determined as echinomycin (1) derivatives containing a 3-hydoxyquinaldic acid molecule in place of one of the two quinoxaline-2-carboxylic acid chromophores. Quinomycins H1 (2) and H2 (3) showed selective cytotoxicity against RG-E1-4 cells bearing the adenovirus oncogenes with IC50s of 11 nM and 12 nM, respectively.