Lyn Kinase Suppresses the Transcriptional Activity of IRF5 in the TLR-MyD88 Pathway to Restrain the Development of Autoimmunity.

Interferon regulatory factor-5 (IRF5), a transcription factor critical for the induction of innate immune responses, contributes to the pathogenesis of the autoimmune disease systemic lupus erythematosus (SLE) in humans and mice. Lyn, a Src family kinase, is also implicated in human SLE, and Lyn-deficient mice develop an SLE-like disease. Here, we found that Lyn physically interacted with IRF5 to inhibit ubiquitination and phosphorylation of IRF5 in the TLR-MyD88 pathway, thereby suppressing the transcriptional activity of IRF5 in a manner independent of Lyn's kinase activity. Conversely, Lyn did not inhibit NF-κB signaling, another major branch downstream of MyD88. Monoallelic deletion of Irf5 alleviated the hyperproduction of cytokines in TLR-stimulated Lyn(-/-) dendritic cells and the development of SLE-like symptoms in Lyn(-/-) mice. Our results reveal a role for Lyn as a specific suppressor of the TLR-MyD88-IRF5 pathway and illustrate the importance of fine-tuning IRF5 activity for the maintenance of immune homeostasis.

Report of the international conference on regulatory endeavors towards the sound development of human cell therapy products.

The regulation of human cell therapy products is a key factor in their development and use to treat human diseases. In that regard, there is a recognized need for a global effort to develop a set of common principles that may serve to facilitate a convergence of regulatory approaches to ensure the smooth and efficient evaluation of products. This conference, with experts from regulatory agencies, industry, and academia, contributed to the process of developing such a document. Elements that could form a minimum consensus package of requirements for evaluating human cell therapy products were the overall focus of the conference. The important regulatory considerations that are unique to human cell therapy products were highlighted. Sessions addressed specific points that are different from those of traditional biological/biotechnological protein products. Panel discussions complemented the presentations. The conference concluded that most of the current regulatory framework is appropriate for cell therapy, but there are some areas where the application of the requirements for traditional biologicals is inappropriate. In addition, it was agreed that there is a need for international consensus on core regulatory elements, and that one of the major international organizations should take the lead in formulating such a consensus document.

In vitro activity of E1210, a novel antifungal, against clinically important yeasts and molds.

E1210 is a new antifungal compound with a novel mechanism of action and broad spectrum of antifungal activity. We investigated the in vitro antifungal activities of E1210 compared to those of fluconazole, itraconazole, voriconazole, amphotericin B, and micafungin against clinical fungal isolates. E1210 showed potent activities against most Candida spp. (MIC(90) of ≤0.008 to 0.06 µg/ml), except for Candida krusei (MICs of 2 to >32 µg/ml). E1210 showed equally potent activities against fluconazole-resistant and fluconazole-susceptible Candida strains. E1210 also had potent activities against various filamentous fungi, including Aspergillus fumigatus (MIC(90) of 0.13 µg/ml). E1210 was also active against Fusarium solani and some black molds. Of note, E1210 showed the greatest activities against Pseudallescheria boydii (MICs of 0.03 to 0.13 µg/ml), Scedosporium prolificans (MIC of 0.03 µg/ml), and Paecilomyces lilacinus (MICs of 0.06 µg/ml) among the compounds tested. The antifungal action of E1210 was fungistatic, but E1210 showed no trailing growth of Candida albicans, which has often been observed with fluconazole. In a cytotoxicity assay using human HK-2 cells, E1210 showed toxicity as low as that of fluconazole. Based on these results, E1210 is likely to be a promising antifungal agent for the treatment of invasive fungal infections.

Efficacy of oral E1210, a new broad-spectrum antifungal with a novel mechanism of action, in murine models of candidiasis, aspergillosis, and fusariosis.

E1210 is a first-in-class, broad-spectrum antifungal with a novel mechanism of action-inhibition of fungal glycosylphosphatidylinositol biosynthesis. In this study, the efficacies of E1210 and reference antifungals were evaluated in murine models of oropharyngeal and disseminated candidiasis, pulmonary aspergillosis, and disseminated fusariosis. Oral E1210 demonstrated dose-dependent efficacy in infections caused by Candida species, Aspergillus spp., and Fusarium solani. In the treatment of oropharyngeal candidiasis, E1210 and fluconazole each caused a significantly greater reduction in the number of oral CFU than the control treatment (P < 0.05). In the disseminated candidiasis model, mice treated with E1210, fluconazole, caspofungin, or liposomal amphotericin B showed significantly higher survival rates than the control mice (P < 0.05). E1210 was also highly effective in treating disseminated candidiasis caused by azole-resistant Candida albicans or Candida tropicalis. A 24-h delay in treatment onset minimally affected the efficacy outcome of E1210 in the treatment of disseminated candidiasis. In the Aspergillus flavus pulmonary aspergillosis model, mice treated with E1210, voriconazole, or caspofungin showed significantly higher survival rates than the control mice (P < 0.05). E1210 was also effective in the treatment of Aspergillus fumigatus pulmonary aspergillosis. In contrast to many antifungals, E1210 was also effective against disseminated fusariosis caused by F. solani. In conclusion, E1210 demonstrated consistent efficacy in murine models of oropharyngeal and disseminated candidiasis, pulmonary aspergillosis, and disseminated fusariosis. These data suggest that further studies to determine E1210's potential for the treatment of disseminated fungal infections are indicated.

Genetic and chemical inhibition of IRF5 suppresses pre-existing mouse lupus-like disease.

The transcription factor IRF5 has been implicated as a therapeutic target for the autoimmune disease systemic lupus erythematosus (SLE). However, IRF5 activation status during the disease course and the effects of IRF5 inhibition after disease onset are unclear. Here, we show that SLE patients in both the active and remission phase have aberrant activation of IRF5 and interferon-stimulated genes. Partial inhibition of IRF5 is superior to full inhibition of type I interferon signaling in suppressing disease in a mouse model of SLE, possibly due to the function of IRF5 in oxidative phosphorylation. We further demonstrate that inhibition of IRF5 via conditional Irf5 deletion and a newly developed small-molecule inhibitor of IRF5 after disease onset suppresses disease progression and is effective for maintenance of remission in mice. These results suggest that IRF5 inhibition might overcome the limitations of current SLE therapies, thus promoting drug discovery research on IRF5 inhibitors.

Synthesis and evaluation of novel antifungal agents-quinoline and pyridine amide derivatives.

Quinoline amide, azaindole amide and pyridine amides were synthesized and tested for in vitro antifungal activity against fungi. These synthesized amides have potent antifungal activity against Candida albicans and Aspergillus fumigatus. Our results suggest that hetero ring amides may be potent antifungal agents that operate by inhibiting the function of Gwt1 protein in the GPI biosynthetic pathway.

Targeting the Golgi apparatus to overcome acquired resistance of non-small cell lung cancer cells to EGFR tyrosine kinase inhibitors.

Epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (EGFR-TKIs) were demonstrated to provide survival benefit in patients with non-small cell lung cancer (NSCLC) harboring activating mutations of EGFR; however, emergence of acquired resistance to EGFR-TKIs has been shown to cause poor outcome. To overcome the TKI resistance, drugs with different mode of action are required. We previously reported that M-COPA (2-methylcoprophilinamide), a Golgi disruptor, suppressed the growth of gastric cancers overexpressing receptor tyrosine kinases (RTKs) such as hepatocyte growth factor receptor (MET) via downregulating their cell surface expression. In this study, we examined the antitumor effect of M-COPA on NSCLC cells with TKI resistance. As a result, M-COPA effectively downregulated cell surface EGFR and its downstream signals, and finally exerted in vivo antitumor effect in NSCLC cells harboring secondary (T790M/del19) and tertiary (C797S/T790M/del19) mutated EGFR, which exhibit acquired resistance to first- and third generation EGFR-TKIs, respectively. M-COPA also downregulated MET expression potentially involved in the acquired resistance to EGFR-TKIs via bypassing the EGFR pathway blockade. These results provide the first evidence that targeting the Golgi apparatus might be a promising therapeutic strategy to overcome the vicious cycle of TKI resistance in EGFR-mutated NSCLC cells via downregulating cell surface RTK expression.

Sulfonamide derivative, E7820, is a unique angiogenesis inhibitor suppressing an expression of integrin alpha2 subunit on endothelium.

In the process of angiogenesis, endothelial adhesion molecules play a significant role in vascular morphogenesis, in coordination with angiogenic factor signaling. Here we report that a novel angiogenesis inhibitor, E7820 (an aromatic sulfonamide derivative), inhibited in vitro proliferation and tube formation of human umbilical vascular endothelial cell (HUVEC). E7820 decreased integrin alpha2, 3, 5, and beta1 in confluent culture of HUVEC, and integrin alpha2 was initially suppressed in mRNA level, followed by decrement of integrins alpha3, 5, and beta1. The inhibition of integrin alpha2 expression in HUVEC showed dose dependence but did not alter the level of CD31. Up-regulation of integrin alpha2 by phorbol 12-myristate 13-acetate abrogated the inhibitory effect of E7820 on tube formation within type I collagen gel, whereas addition of antibody against integrin alpha2 canceled the phorbol 12-myristate 13-acetate effect. These results suggest that E7820 inhibited tube formation through the suppression of integrin alpha2. Oral administration of E7820 remarkably resulted in inhibition of tumor-induced angiogenesis in mouse dorsal air sac model, and tumor growth of human colorectal tumor cell lines (WiDr and LoVo) was inhibited in xenotransplanted model in mice. This is the first time that a small molecule has been shown to modulate integrins, and this finding may provide the basis for a new approach to antiangiogenic therapy through the suppression of integrin alpha2 on endothelium.

M-COPA, a Golgi Disruptor, Inhibits Cell Surface Expression of MET Protein and Exhibits Antitumor Activity against MET-Addicted Gastric Cancers.

The Golgi apparatus is responsible for transporting, processing, and sorting numerous proteins in the cell, including cell surface-expressed receptor tyrosine kinases (RTK). The small-molecule compound M-COPA [2-methylcoprophilinamide (AMF-26)] disrupts the Golgi apparatus by inhibiting the activation of Arf1, resulting in suppression of tumor growth. Here, we report an evaluation of M-COPA activity against RTK-addicted cancers, focusing specifically on human gastric cancer (GC) cells with or without MET amplification. As expected, the MET-addicted cell line MKN45 exhibited a better response to M-COPA than cell lines without MET amplification. Upon M-COPA treatment, cell surface expression of MET was downregulated with a concurrent accumulation of its precursor form. M-COPA also reduced levels of the phosphorylated form of MET along with the downstream signaling molecules Akt and S6. Similar results were obtained in additional GC cell lines with amplification of MET or the FGF receptor FGFR2 MKN45 murine xenograft experiments demonstrated the antitumor activity of M-COPA in vivo Taken together, our results offer an initial preclinical proof of concept for the use of M-COPA as a candidate treatment option for MET-addicted GC, with broader implications for targeting the Golgi apparatus as a novel cancer therapeutic approach. Cancer Res; 76(13); 3895-903. ©2016 AACR.

Alpha6beta1 integrin induces proteasome-mediated cleavage of erbB2 in breast cancer cells.

ErbB2 and alpha6 integrin have been implicated in malignancy of breast cancer cells. Here we have determined the influence of alpha6beta1 integrin on erbB2 signaling in anchorage-independent growth, using MDA-MB435 breast cancer cells. Firstly, we transfected the cells with erbB2 cDNA, and isolated cells with high or low levels of alpha6beta1 integrin by cell sorting (alpha6H-ErbB and alpha6L-ErbB). We found that an erbB ligand, heregulin beta1, enhanced growth activity of alpha6L-ErbB cells, but not alpha6H-ErbB cells. Secondly, we established cells expressing a beta4 integrin deletion mutant (beta4-deltacyt), which selectively inhibited alpha6beta1 integrin expression and adhesion to laminin-1. Again, heregulin beta1 enhanced the growth of erbB2 cDNA-transfected beta4-deltacyt cells, but not mock cells. Western blot analysis revealed that heregulin beta1 stimulated phosphorylation of Akt and its downstream molecules, GSK3beta and p70S6kinase, and that the extent of phosphorylation was greater in ErbB2/beta4-deltacyt cells than ErbB2/mock cells. Furthermore, we found that the erbB2 cytoplasmic domain was truncated in ErbB2/mock cells, which was independent of ligand stimulation and adhesion, and was suppressed by proteasome inhibitors. These results suggest that alpha6beta1 integrin inhibits erbB2 signals by inducing proteasome-dependent proteolytic cleavage of the erbB2 cytoplasmic domain, and may thereby contribute to the regulation of tumor growth.

An angiogenesis inhibitor E7820 shows broad-spectrum tumor growth inhibition in a xenograft model: possible value of integrin alpha2 on platelets as a biological marker.

We reported previously that an angiogenesis inhibitor, E7820, inhibits in vitro tube formation of human umbilical vein endothelial cell through the suppression of integrin alpha2 expression. Here we describe the antiangiogenic and antitumor effects of E7820 in mice and discuss the feasibility of using platelet integrin alpha2 expression on platelets as a biological marker of the efficacy of E7820. Oral administration of E7820 significantly inhibited basic fibroblast growth factor-induced angiogenesis in Matrigel implants and human colon WiDr tumor-induced angiogenesis in a dorsal air sac model. Twice-daily treatment with E7820 clearly inhibited the s.c. tumor growth of seven tumor cell lines derived from human colon, breast, pancreas, and kidney, and completely suppressed the growth of human pancreatic KP-1 and human colon LoVo cell lines. Moreover, E7820 significantly inhibited the growth of KP-1 and human colon tumor Colo320DM cells orthotopically implanted in the pancreas and cecum, respectively. The efficacy of E7820 was comparable in the s.c. and orthotopic transplantation models. Immunohistochemical analyses using anti-CD31 antibody showed that E7820 significantly reduced microvessel density in orthotopically implanted KP-1 tumor. E7820 reduced integrin alpha2 expression on a megakaryocytic cell line, Dami cells, induced by phorbol 12-myristate 13-acetate treatment. It also decreased the expression level of integrin alpha2 on platelets withdrawn from mice bearing s.c. KP-1 tumor at a dosage close to that affording antitumor activity. These data demonstrate that E7820 showed a broad-spectrum antitumor effect in mice through inhibition of angiogenesis and indicate that the decrease of integrin alpha2 on platelets might serve as a biological marker for the antitumor efficacy of E7820.

Effects of ER-37328 on primary tumor, liver metastasis, and life span in a murine colon 38 orthotopic transplantation model.

12,13-Dihydro-5-[2-(dimethylamino)ethyl]-4H-benzo[c] pyrimido[5,6,1-jk] carbazole-4,6,10(5H,11H)-trione hydrochloride (ER-37328) is a novel topoisomerase II poison with potent tumoricidal activity against solid tumor cells both in vitro and in vivo. Here, we describe studies on the effects of ER-37328 on the primary tumor, liver metastasis, and survival in a murine Colon 38 orthotopic transplantation model. When ER-37328 (10 mg/kg) was administered i.v. at 11 days or 20 days after transplantation, strong regression of the primary tumor was observed on both administration schedules. On the later schedule, ER-37328 completely blocked liver metastasis, whereas the mean number of metastases in the control group was 23.9. To examine the antitumor activity against Colon 38 at the liver in more detail, ER-37328 was administered to mice that had received an inoculation of Colon 38 tumor into the liver. ER-37328 showed strong tumor-regression activity against Colon 38 growing in the liver. In addition, administration of ER-37328 on a schedule of every 7 days four times caused a significant increase of 79% in life span in the orthotopic transplantation model, calculated by using mean survival times. Pharmacokinetic study revealed that ER-37328 was highly distributed to the tumor and organs. The ratios of the area under the concentration-time curves of ER-37328 in the tumor, lung, liver, and kidney versus plasma were 81, 77, 47, and 40, respectively. This high distribution to the tumor and liver may explain the potent antitumor activity of ER-37328 against Colon 38 tumor in the liver. In conclusion, the topoisomerase II poison ER-37328 is a promising candidate for clinical application against colon cancer.

Antitumor activity of ER-37328, a novel carbazole topoisomerase II inhibitor.

DNA topoisomerase II has been shown to be an important therapeutic target in cancer chemotherapy. Here, we describe studies on the antitumor activity of a novel topoisomerase II inhibitor, ER-37328 [12,13-dihydro-5-[2-(dimethylamino)ethyl]-4H-benzo[c]pyrimido[5,6,1- jk]carbazole-4,6,10(5H,11H)-trione hydrochloride]. ER-37328 inhibited topoisomerase II activity at 10 times lower concentration than etoposide in relaxation assay and induced double-strand DNA cleavage within 1 h in murine leukemia P388 cells, in a bell-shaped manner with respect to drug concentration. The maximum amount of DNA cleavage was obtained at 2 microM. Like etoposide, ER-37328 (2 microM) induced topoisomerase II-DNA cross-linking in P388 cells. A spectroscopic study of ER-37328 mixed with DNA demonstrated that ER-37328 has apparent binding activity to DNA. ER-37328 showed potent growth-inhibitory activity against a panel of 21 human cancer cell lines [mean (50% growth-inhibitory concentration) GI50 = 59 nM]. COMPARE analysis according to the National Cancer Institute screening protocol showed that the pattern of the growth-inhibitory effect of ER-37328 was similar to that of etoposide, but different from that of doxorubicin. Studies on etoposide-, amsacrine [4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA)]-, and camptothecin-resistant P388 cell lines showed that: (a) etoposide- and m-AMSA-resistant P388 cell lines were partially resistant to ER-37328 compared with the parental cell line; and (b) a camptothecin-resistant cell line showed no cross-resistance to ER-37328. In addition, ER-37328 overcame P-glycoprotein-mediated resistance. In vivo, ER-37328 produced potent tumor regression of Colon 38 carcinoma inoculated s.c., and its activity was superior to that of etoposide or doxorubicin. These results indicate that ER-37328 inhibits topoisomerase II activity through the formation of topoisomerase II-DNA cleavable complex and has potent antitumor activity both in vitro and in vivo.

Profiling novel sulfonamide antitumor agents with cell-based phenotypic screens and array-based gene expression analysis.

A series of small molecules from sulfonamide-focused libraries have been evaluated in these laboratories to discover novel antitumor agents. Cell-based screens using flow cytometric analysis revealed the presence of two distinct classes of cell cycle inhibitors in this series; one (including E7010 and ER-67865) arrested mitosis by preventing tubulin polymerization; and the other (including E7070 and ER-68487) caused a decrease in the S-phase fraction along with cell cycle perturbation in G1 and/or G2 via an unknown mechanism(s). To further characterize both classes of antitumor sulfonamides with respect to their effects on gene expression, we used oligonucleotide microarray analysis for representative compounds. Consistent with the phenotypic observations, essentially the same transcription profiles were found between E7010 and ER-67865 and also between E7070 and ER-68487. However, there was very little overlap between genes affected by E7010 and E7070. As a characteristic expression change for microtubule-depolymerizing agents, the down-regulation of alpha-tubulin transcripts was evident in both E7010- and ER-67865-treated cells. On the other hand, E7070 and ER-68487 repressed significantly the expression of a variety of genes involved in metabolic processes, cell cycle progression, immune response, and signal transduction. Of the compounds examined, E7010 and E7070 have progressed to clinical trials, demonstrating some objective responses in the Phase I setting. Described herein is profiling of novel anticancer drug candidates from the sulfonamide class based on phenotypic screens and gene expression analysis. This includes a translational research that may suggest potentially useful markers for pharmacodynamic drug assessment in clinic.

Array-based structure and gene expression relationship study of antitumor sulfonamides including N-[2-[(4-hydroxyphenyl)amino]-3-pyridinyl]-4-methoxybenzenesulfonamide and N-(3-chloro-7-indolyl)-1,4-benzenedisulfonamide.

Compounds from sulfonamide-focused libraries have been evaluated in cell-based antitumor screens using the COMPARE analysis with a panel of 39 human cancer cell lines and flow cytometric cell cycle analysis. Thus far, 2 (N-[2-[(4-hydroxyphenyl)amino]-3-pyridinyl]-4-methoxybenzenesulfonamide (E7010)) and 3 (N-(3-chloro-7-indolyl)-1,4-benzenedisulfonamide (E7070)) have been selected from the collections as potent cell cycle inhibitors, which have progressed to clinical trials. Compound 2 is an orally active antimitotic agent disrupting tubulin polymerization, whereas compound 3 belongs to a novel class of antiproliferative agents causing a decrease in the S phase fraction along with G1 and/or G2 accumulation in various cancer cell lines. Because both compounds exhibited preliminary clinical activities in the phase I setting, we decided to examine further this series of oncolytic small molecules, particularly by using high-density oligonucleotide microarray analysis. The array data have enabled us to characterize these two classes of antitumor sulfonamides on the basis of gene expression changes, illuminating the essential pharmacophore structure and drug-sensitive cellular pathways for each class. Moreover, the dual character of 5 (N-(3-chloro-7-indolyl)-4-methoxybenzenesulfonamide (ER-67880)), resembling both 2 and 3, was revealed by array-based transcription profiling, though the 3-type profile of this molecule had not been apparent in the cell-based phenotypic screens. These results provide an example of the utility of structure and gene expression relationship studies in medicinal genomics.

Aberrant expression of integrin and erbB subunits in breast cancer cell lines.

Integrin and growth factor receptors play an important role in cell functions and their aberrant expressions are implicated in breast cancer malignancy. Recent studies have shown that integrins physically and functionally associate with growth factor receptors suggesting the cooperative regulation of these two signals. We studied the expression of integrin and erbB subunits by flow cytometer in human normal mammary epithelial (HME) cell, non-metastatic (MCF-7, ZR-75-1, MDA-MB453) and metastatic tumor cell lines (MDA-MB231, MDA-MB435). Compared with HME cells, all of non-metastatic and metastatic cell lines showed decreased expressions of alpha2 and beta4 integrin subunits. Two metastatic cell lines, but not three non-metastatic tumor cell lines, expressed alpha5 and alpha6 comparable to HME cells. There was no correlation of erbB2 expression with integrin expressions. We isolated MDA-MB435 subpopulations expressing lower amount of alpha6 integrin and found that alpha5, but not alpha2 and alphav integrins, was concomitantly decreased while erbB family was not affected. Then we transfected erbB2 gene into MDA-MB435 and found the induction of erbB3 expression but not erbB1 and erbB4. However, erbB2 transfection had no effect on the expression of alpha6 and beta4 integrin subunits. These data suggest that the expression of alpha5 and alpha6 integrins may contribute to metastasis, and that the regulation of erbB2 and alpha6 integrin expressions is independent in breast cancer cells.

Pladienolides, new substances from culture of Streptomyces platensis Mer-11107. III. In vitro and in vivo antitumor activities.

We have discovered seven novel 12-membered macrolides, pladienolides A to G, from Streptomyces platensis Mer-11107, with pladienolide B the most potently inhibiting hypoxia induced-VEGF expression and proliferation of the U251 cancer cell line. A growth inhibitory study using a 39-cell line drug-screening panel demonstrated that pladienolide B has strong antitumor activities in vitro. A COMPARE analysis reveals that it has a unique antitumor spectrum that sets it apart from anticancer drugs currently in clinical use. This result suggests that pladienolide B has a novel mechanism of action. A series of xenograft studies were conducted to evaluate the in vivo potency of pladienolides. Pladienolide B extensively inhibited tumor growth in xenograft models. In the most sensitive model, using BSY-1 xenografts, tumors were completely regressed by administration of pladienolide B. For the reason of their novel mechanism of action and excellent in vivo efficacy, pladienolides appear to have major potential for use in cancer treatment.

Lenvatinib, an angiogenesis inhibitor targeting VEGFR/FGFR, shows broad antitumor activity in human tumor xenograft models associated with microvessel density and pericyte coverage.

BACKGROUND: Lenvatinib is an oral inhibitor of multiple receptor tyrosine kinases (RTKs) targeting vascular endothelial growth factor receptor (VEGFR1-3), fibroblast growth factor receptor (FGFR1-4), platelet growth factor receptor α (PDGFR α), RET and KIT. Antiangiogenesis activity of lenvatinib in VEGF- and FGF-driven angiogenesis models in both in vitro and in vivo was determined. Roles of tumor vasculature (microvessel density (MVD) and pericyte coverage) as biomarkers for lenvatinib were also examined in this study. METHOD: We evaluated antiangiogenesis activity of lenvatinib against VEGF- and FGF-driven proliferation and tube formation of HUVECs in vitro. Effects of lenvatinib on in vivo angiogenesis, which was enhanced by overexpressed VEGF or FGF in human pancreatic cancer KP-1 cells, were examined in the mouse dorsal air sac assay. We determined antitumor activity of lenvatinib in a broad panel of human tumor xenograft models to test if vascular score, which consisted of high MVD and low pericyte coverage, was associated with sensitivity to lenvatinib treatment. Vascular score was also analyzed using human tumor specimens with 18 different types of human primary tumors. RESULT: Lenvatinib inhibited VEGF- and FGF-driven proliferation and tube formation of HUVECs in vitro. In vivo angiogenesis induced by overexpressed VEGF (KP-1/VEGF transfectants) or FGF (KP-1/FGF transfectants) was significantly suppressed with oral treatments of lenvatinib. Lenvatinib showed significant antitumor activity in KP-1/VEGF and five 5 of 7 different types of human tumor xenograft models at between 1 to 100 mg/kg. We divided 19 human tumor xenograft models into lenvatinib-sensitive (tumor-shrinkage) and relatively resistant (slow-growth) subgroups based on sensitivity to lenvatinib treatments at 100 mg/kg. IHC analysis showed that vascular score was significantly higher in sensitive subgroup than relatively resistant subgroup (p < 0.0004). Among 18 types of human primary tumors, kidney cancer had the highest MVD, while liver cancer had the lowest pericyte coverage, and cancers in Kidney and Stomach had highest vascular score. CONCLUSION: These results indicated that Lenvatinib inhibited VEGF- and FGF-driven angiogenesis and showed a broad spectrum of antitumor activity with a wide therapeutic window. MVD and pericyte-coverage of tumor vasculature might be biomarkers and suggest cases that would respond for lenvatinib therapy.

Therapeutic potential and molecular mechanism of a novel sulfonamide anticancer drug, indisulam (E7070) in combination with CPT-11 for cancer treatment.

PURPOSE: Indisulam (N-(-3-chloro-7-indolyl)-1,4-benzenedisulfonamide; E7070) is an experimental anticancer agent. Microarray analysis indicates that indisulam downregulates several genes involved in drug resistance, and this finding led us to test the effect of combining indisulam with other anticancer drugs. We investigated the antitumor effect and mechanism of synergism when indisulam was administered in combination with CPT-11. METHODS: In vitro cytotoxic activity was examined using a cell counter kit, and the combination effect was determined by isobologram analysis. The level of topoisomerase IIα was measured by Western blotting. The in vivo antitumor effect was assessed in mice inoculated with human colorectal cancer SW620 cells. RESULTS: Isobologram analysis indicated that a 24-h exposure to indisulam and SN-38, an active metabolite of CPT-11, had a synergistic effect in HCT116 and SW620 cells and an additive effect in HCT15 and WiDr cells. Prolongation of exposure to 48 h resulted in a synergistic effect in HCT15 and WiDr cells. Treatment with SN-38 alone increased the amount of intracellular topoisomerase IIα in all cell lines tested. Co-treatment with indisulam suppressed the SN-38-induced upregulation of topoisomerase IIα after 24 h of exposure in HCT116 and SW620 cells and after 48 h of exposure in HCT15 and WiDr cells. This apparent association between a synergistic effect and suppression of SN-38-mediated upregulation of topoisomerase IIα suggests that indisulam enhances SN-38 cytotoxicity by suppressing topoisomerase IIα upregulation to compensate for topoisomerase I inhibition by SN-38. Synergy was also observed in xenografted tumors and was accompanied by complete suppression of topoisomerase IIα upregulation induced by CPT-11 treatment. CONCLUSION: These observations prompted the clinical evaluation of indisulam and CPT-11 combination therapy.

Stem cell factor/c-kit signaling promotes the survival, migration, and capillary tube formation of human umbilical vein endothelial cells.

c-kit receptor tyrosine kinase is a marker of progenitor cells, which differentiate into blood and/or vascular endothelial cells, and has an important role in the amplification/mobilization of progenitor cells. c-kit is expressed in mature endothelial cells, but its role there is unclear. Stem cell factor, a c-kit ligand, dose-dependently promoted survival, migration, and capillary tube formation of human umbilical vein endothelial cells. These effects mimicked those of vascular endothelial growth factor, except that stem cell factor did not sufficiently support proliferation of these cells. After exposing cells to this factor, Akt, Erk1/2, and c-kit were immediately (