Inhibitors of insulin-like growth factor-1 receptor tyrosine kinase are preferentially cytotoxic to nutrient-deprived pancreatic cancer cells.

Chronic deprivation of nutrients is rare in normal tissues, however large areas of tumor are nutrient-starved and hypoxic due to a disorganized vascular system. Some cancers show an inherent ability to tolerate severe growth conditions. Therefore, we screened chemical compounds to identify cytotoxic agents that function preferentially in nutrient-deprived conditions. We found that AG1024, a specific inhibitor of insulin-like growth factor-1 receptor tyrosine kinase (IGF-1R), showed preferential cytotoxicity to human pancreatic cancer cells in nutrient-deprived conditions relative to cells in nutrient-sufficient conditions. The cytotoxicity of I-OMe-AG538 (another specific inhibitor of IGF-1R kinase) was also enhanced in nutrient-deprived cells. In addition, AG1024 and I-OMe-AG538 potently inhibited IGF-1R activation to nutrient-deprived cells. In contrast, conventional chemotherapeutic drugs, as well as inhibitors of PDGFR and EGFR kinases, elicited weak cytotoxicity. These data indicate that nutrient-deprived human pancreatic cancer cells have increased sensitivity to inhibition of IGF-1R activation. IGF-1R inhibitors offer a promising strategy for anticancer therapeutic approaches that are oriented toward tumor microenvironment.

Rakicidin A: a hypoxia-selective cytotoxin.

Hypoxia is a common feature of many solid tumors and contributes to their progression. Hypoxic cells in the tumor are not only involved in therapeutic resistance to chemotherapy and radiotherapy but are also relevant to tumor angiogenesis. To identify novel hypoxia-selective cytotoxins, we screened 20000 cultured broths of microorganisms and found that rakicidin A showed significant hypoxia-selective cytotoxicity. Rakicidin A was approximately 17.5-fold more cytotoxic under hypoxic than under normoxic conditions. CoCl2 and antioxidants had no effect on the rakicidin A cytotoxicity under normoxic conditions and rakicidin A did not show the inhibitory effects on HIF-1 transcriptional activity under hypoxic conditions. Thus, although the action mechanism of the hypoxia-selective cytotoxicity of rakicidin A was unknown, our screening study suggested that rakicidin A acts as an antitumor agent for selective therapy against solid tumors.

Anthracyclines, small-molecule inhibitors of hypoxia-inducible factor-1 alpha activation.

Hypoxia-inducible factor-1 (HIF-1) is a central mediator of cellular responses to low oxygen and has recently become an important therapeutic target for solid tumor therapy. To identify small molecule inhibitors of the HIF-1 transcriptional activation, we have established a high through-put assay system using a stable transformant of mammalian cells that express the luciferase reporter gene construct containing a HIF-1 binding site. Using this system, we screened 5000 cultured broths of microorganisms, and we found that cinerubin (1-hydroxy aclacinomycin B) showed a significant inhibition of the reporter activity induced by hypoxic conditions. In addition, we demonstrated that aclarubicin also inhibited the HIF-1 transcriptional activity under hypoxic conditions, but neither doxorubicin nor daunorubicin inhibited it. Consistent with these results, cinerubin and aclarubicin inhibited the hypoxic induction of the vascular endothelial growth factor (VEGF) protein in HepG2 cells, but neither doxorubicin nor daunorubicin affected it. Thus, our results suggested that some anthracyclines are also acting as angiogenesis inhibitors.

Augmentation of cellular immunity by kigamicin D.

Kigamicin D did not show any immunosuppressive activity in mixed lymphocyte culture reaction and mitogen induced lymphocyte blastogenesis in vitro and graft versus host reaction in vivo. Natural killer cell activity in spleen cells was not affected by oral administration of kigamicin D. Instead, delayed-type hypersensitivity response to sheep red blood cells was stimulated at a broad dosage level. It is concluded that kigamicin D increases cellular immunity to specific antigen.

Antitumor effect of kigamicin D on mouse tumor models.

Kigamicin D is a novel anticancer agent that was identified using a new screening strategy that targets the tolerance of cancer cells to nutrient starvation [1, 2]. Oral administration of kigamicin D was previously described to show a strong antitumor effect in human tumor xenograft models of pancreatic tumors [2]. In this paper we describe that kigamicin D shows the same selective cytotoxicity against normal human cells such as lung fibroblast and prostate stromal cells under nutrient starved condition as against cancer cells. Kigamicin D inhibited tumor cell-induced angiogenesis in a dorsal air sac assay. On the basis of these results we tested other human tumor xenograft models and transplantable syngeneic tumor models in order to determine the spectrum of activity of kigamicin D against various cancers. Kigamicin D showed a weak antitumor effect against LX-1 and DMS-273 lung cancers, but had no effect on DLD-1 colon cancers. When tested against syngeneic tumors, kigamicin D showed a weak antitumor effect against colon26, but showed augmentation of tumor growth on IMC carcinoma at a broad dosage level. Kigamicin D does not show good antitumor activity against human xenograft tumors except pancreatic tumors and murine syngeneic tumors. We found that kigamicin D has excellent antitumor effect specific to pancreatic cancers. Surprisingly, high dosage of kigamicin D increased tumor growth of IMC carcinoma by than 200%. The phenomenon suggests that kigamicin D may cause some immunological response to the tumor.

Absolute configuration of kigamicins A, C and D.

The stereochemistry of kigamicins A (1), C (2) and D (3) were elucidated by a combination of X-ray crystallographic analysis and degradation studies. The absolute structures of kigamicins thus determined were depicted as shown in Fig. 2.

Kigamicin D, a novel anticancer agent based on a new anti-austerity strategy targeting cancer cells' tolerance to nutrient starvation.

Both tolerance to nutrient starvation and angiogenesis are essential for cancer progression because of the insufficient supply of nutrients to tumor tissue. Since chronic nutrient starvation seldom occurs in normal tissue, cancer's tolerance to nutrient starvation should provide a novel target for cancer therapy. In this study, we propose an anti-austerity strategy to exploit the ability of agents to eliminate cancer cells' tolerance to nutrient starvation. We established a simple screening method for agents that inhibit cancer cell viability preferentially during nutrient starvation, using PANC-1 cell line cultured in nutrient-rich and nutrient-deprived media. After screening over 2000 culture media of actinomycetes, we identified a new compound, kigamicin D (C(48)H(59)NO(19)), which shows preferential cytotoxicity to cancer cells under nutrient-deprived conditions, but hardly any cytotoxicity under nutrient-rich conditions. Both subcutaneous and oral administration of kigamicin D strongly suppressed the tumor growth of several tested pancreatic cancer cell lines in nude mice. Moreover, kigamicin D was observed to block the activation of Akt induced by nutrient starvation. Therefore, our results suggest that kigamicin D be a candidate for implementing our novel concept, anti-austerity, which may serve as a new strategy for cancer therapy.

Specific inhibition of hypoxia-inducible factor (HIF)-1 alpha activation and of vascular endothelial growth factor (VEGF) production by flavonoids.

Screening using a reporter under the control of the hypoxia-response element (HRE) identified several flavonoids and homoisoflavonoids that inhibit the activation of HRE under hypoxic conditions. Among various compounds, isorhamnetin, luteolin, quercetin, and methyl ophiopogonanone B (MOB) were effective at 3 to 9 microg/ml in inhibiting the reporter activity. The expression of vascular endothelial growth factor (VEGF) mRNA during hypoxia was also inhibited by MOB in HepG2 cells, but the effective doses were 10 to 20 microg/ml. MOB caused destabilization of hypoxia-inducible factor (HIF)-1alpha, as revealed by Western blotting, that was dependent on proteasome activity and the tumor suppressor, p53. The tubular formation and migration of human umbilical vein endothelial cells was also inhibited by MOB. MOB is expected to act as an inhibitor of angiogenesis.

HIF-1-dependent VEGF reporter gene assay by a stable transformant of CHO cells.

For the establishment of a screening system to detect inhibitors of vascular endotherial growth factor (VEGF) expression, a stable transformant of Chinese hamster ovary cells was isolated and cloned by transfection of a hypoxia-inducible factor 1 (HIF-1)-dependent VEGF promoter reporter gene. The expression of the reporter gene in the clone cells, as measured by luciferase activity, was stable. Hypoxic responses were best observed at an initial cell density of 2 x 10(4)/well. The maximal increase of luciferase activity was 30 fold. In the highest cell density of 8 x 10(4)/well (2.1 x 10(5)/cm(2)), basal activity was increased 13-15 fold compared to that at the lower cell densities, and did not respond to hypoxia. Addition of CoCl(2), which is known to mimic hypoxia, increased luciferase activity more than 10 times in normoxia. Nitric oxide donors, which are known to suppress the activation of HIF-1, inhibited expression of the VEGF promoter reporter gene under hypoxia. Histone deacetylase inhibitors, trichostatin A and sodium n-butyrate which are known to stimulate transcription of many genes enhanced its transcription in hypoxia. These results indicate that the stable transformant is a useful tool for screening of HIF-1 modifiers.

Kigamicins, novel antitumor antibiotics. I. Taxonomy, isolation, physico-chemical properties and biological activities.

Novel antibiotics named kigamicin A, B, C, D, and E were discovered from the culture broth of Amycolatopsis sp. ML630-mF1 by their selective killing activity against PANC-1 cells only under a nutrient starved condition. Under a condition of nutrient starvation, kigamicins A, B, C, and D inhibited PANC-1 cell survival at 100 times lower concentration than in normal culture. Kigamicins showed antimicrobial activity against Gram-positive bacteria including methicillin resistant Staphylococcus aureus (MRSA). Kigamicin D inhibited the growth of various mouse tumor cell lines at IC50 of about 1 microg/ml.

Kigamicins, novel antitumor antibiotics. II. Structure determination.

Kigamicin A (1), B (2), C (3), D (4) and E (5) are novel antitumor antibiotics. Their structures were determined by spectroscopic analyses including various NMR measurements. Kigamicins have a unique aglycone of fused octacyclic ring system containing seven of six-membered rings and one oxazolidine. The aglycone links a sugar chain composed of one to four deoxysugars. These sugars were found to be amicetose and oleandrose.

Induction of p16/INK4a gene expression and cellular senescence by toyocamycin.

We constructed an assay system of a luciferase reporter with p16/lNK4a gene transcriptional regulatory domain to identify p16-inducing substances, and found toyocamycin to induce gene expression from the screening of culture fluids of Streptomyces. Toyocamycin is a nucleoside analog, and it increased the p16 mRNA level in human normal fibroblasts or synovial cells as assessed by Northern blot hybridization or real time RT-PCR. It also induced cellular senescence in normal human fibroblasts. The transcriptional regulatory regions of human p16 gene that were responsible for the induction were analyzed using deletion mutants of the transcriptional regulatory region of p16 linked to the luciferase gene. The DNA fragment -111 to +1 bp from the cap site was sufficient to drive toyocamycin-activated transcription of p16/luciferase reporter. Nucleotide sequences within this domain contained the Sp1- and Ets-binding sequences. Mutations were introduced into these sequences, and the Sp1 sequence was found to be critical for the induction, and this notion was confirmed from gel-mobility shift assay.