Reforms of regulatory pathways for approval of new antineoplastic drugs in Japan from 2004 to 2019 and accompanying changes in pivotal clinical trial designs.

Background The Japanese Pharmaceuticals and Medical Devices Agency (PMDA) was established in 2004. Since then, various pieces of legislation, notices, and guidelines have been issued, and the regulatory approval pathways for domestic drugs have been diversified. However, the effects of these measures have not been fully examined. We examined the impact of these measures on the approval of antineoplastic drugs and the design of pivotal clinical trials for efficacy assessment by the PMDA. Methods We collected data on the antineoplastic drugs approved by the PMDA in fiscal years 2004-2019. We extracted the approval review pathways and the pivotal clinical trial designs from the PMDA review reports, and analyzed them to identify patterns. Results In total, 387 indications in oncology were approved by the PMDA in fiscal years 2004-2019, or 365 indications excluding multiple regulatory pathways. The number of approved indications generally increased year on year (p < 0.001). The largest number of approved indications was under the Orphan Drug Designation (31%, 114/365) and this continues to increase (p < 0.001). In the 288 indications for which clinical trial data were submitted for review, the pivotal clinical trial designs changed significantly (p < 0.001) after the guideline on clinical evaluation for antineoplastic drugs was revised in 2006. Conclusion The number of indications in oncology approved by the PMDA has been increasing over the past 16 years, alongside changes in regulatory pathways. The 2006 guideline on clinical evaluation had a particular impact on pivotal clinical trial designs.

Lamellarin 14, a derivative of marine alkaloids, inhibits the T790M/C797S mutant epidermal growth factor receptor.

The emergence of acquired resistance is a major concern associated with molecularly targeted kinase inhibitors. The C797S mutation in the epidermal growth factor receptor (EGFR) confers resistance to osimertinib, a third-generation EGFR-tyrosine kinase inhibitor (EGFR-TKI). We report that the derivatization of the marine alkaloid topoisomerase inhibitor lamellarin N provides a structurally new class of EGFR-TKIs. One of these, lamellarin 14, is effective against the C797S mutant EGFR. Bioinformatic analyses revealed that the derivatization transformed the topoisomerase inhibitor-like biological activity of lamellarin N into kinase inhibitor-like activity. Ba/F3 and PC-9 cells expressing the EGFR in-frame deletion within exon 19 (del ex19)/T790M/C797S triple-mutant were sensitive to lamellarin 14 in a dose range similar to the effective dose for cells expressing EGFR del ex19 or del ex19/T790M. Lamellarin 14 decreased the autophosphorylation of EGFR and the downstream signaling in the triple-mutant EGFR PC-9 cells. Furthermore, intraperitoneal administration of 10 mg/kg lamellarin 14 for 17 days suppressed tumor growth of the triple-mutant EGFR PC-9 cells in a mouse xenograft model using BALB/c nu/nu mice. Thus, lamellarin 14 serves as a novel structural backbone for an EGFR-TKI that prevents the development of cross-resistance against known drugs in this class.

Discovery of Inhibitors of Membrane Traffic from a Panel of Clinically Effective Anticancer Drugs.

In addition to their major targets, clinically effective drugs may have unknown off-targets. By identifying such off-targets it may be possible to repurpose approved drugs for new indications. We are interested in the Golgi apparatus as a novel target for cancer therapy, but there is a paucity of candidate Golgi-disrupting drugs. Here, we aimed to identify Golgi-disrupting compounds from a panel of 34 approved anticancer drugs by using HBC-4 human breast cancer cells and immunofluorescence microscopy to visualize the Golgi apparatus. The screen identified five drugs having Golgi-disrupting activity. Four of them were vinca alkaloids (vinorelbine, vindesine, vincristine and vinblastine), and the fifth drug was eribulin. This is the first study to demonstrate that vinorelbine, vindesine and eribulin possess Golgi-disrupting activity. The 5 drugs are known to inhibit tubulin polymerization and to induce microtubule depolymerization. Interestingly, a microtubule-stabilizer paclitaxel did not induce Golgi-disruption, suggesting that the three-dimensionally preserved microtubules are partly responsible for maintaining the Golgi complex. Concerning eribulin, a noteworthy drug because of its high clinical efficacy against advanced breast cancer, we further confirmed its Golgi-disrupting activity in 3 different human breast cancer cell lines, BSY-1, MDA-MB-231 and MCF-7. Golgi-disruption may contribute to anticancer efficacy of eribulin. In conclusion, the present study revealed that 4 vinca alkaloids and eribulin possessed potential Golgi-disrupting activity among a panel of 34 approved anticancer drugs. Other drugs covering various molecular-targeted drugs and classical DNA-damaging drugs showed no Golgi-disrupting effect. These results suggest that tubulin polymerization-inhibitors might be promising candidate drugs with Golgi-disrupting activity.

Family-wide Analysis of the Inhibition of Arf Guanine Nucleotide Exchange Factors with Small Molecules: Evidence of Unique Inhibitory Profiles.

Arf GTPases and their guanine nucleotide exchange factors (ArfGEFs) are major regulators of membrane traffic and organelle structure in cells. They are associated with a variety of diseases and are thus attractive therapeutic targets for inhibition by small molecules. Several inhibitors of unrelated chemical structures have been discovered, which have shown their potential in dissecting molecular pathways and blocking disease-related functions. However, their specificity across the ArfGEF family has remained elusive. Importantly, inhibitory responses in the context of membranes, which are critical determinants of Arf and ArfGEF cellular functions, have not been investigated. Here, we compare the efficiency and specificity of four structurally distinct ArfGEF inhibitors, Brefeldin A, SecinH3, M-COPA, and NAV-2729, toward six ArfGEFs (human ARNO, EFA6, BIG1, and BRAG2 and Legionella and Rickettsia RalF). Inhibition was assessed by fluorescence kinetics using pure proteins, and its modulation by membranes was determined with lipidated GTPases in the presence of liposomes. Our analysis shows that despite the intra-ArfGEF family resemblance, each inhibitor has a specific inhibitory profile. Notably, M-COPA is a potent pan-ArfGEF inhibitor, and NAV-2729 inhibits all GEFs, the strongest effects being against BRAG2 and Arf1. Furthermore, the presence of the membrane-binding domain in Legionella RalF reveals a strong inhibitory effect of BFA that is not measured on its GEF domain alone. This study demonstrates the value of family-wide assays with incorporation of membranes, and it should enable accurate dissection of Arf pathways by these inhibitors to best guide their use and development as therapeutic agents.

M-COPA, a novel Golgi system disruptor, suppresses apoptosis induced by Shiga toxin.

Shiga toxin (Stx) is a main virulence factor of Stx-producing Escherichia coli (STEC) that contributes to diarrhea and hemorrhagic colitis and occasionally to fatal systemic complications. Therefore, the development of an antidote to neutralize Stx toxicity is urgently needed. After internalization into cells, Stx is transferred to the Golgi apparatus via a retrograde vesicular transport system. We report here that 2-methylcoprophilinamide (M-COPA), a compound that induces disassembly of the Golgi apparatus by inactivating ADP-ribosylation factor 1 (Arf1), suppresses Stx-induced apoptosis. M-COPA inhibited transport of Stx from the plasma membrane to the Golgi apparatus and suppressed degradation of anti-apoptotic proteins and the activation of caspases. These findings suggest that inhibition of Stx retrograde transport by M-COPA could be a novel approach to suppress Stx toxicity.

Abdominal Infection Suppresses the Number and Activity of Intrahepatic Natural Killer Cells and Promotes Tumor Growth in a Murine Liver Metastasis Model.

BACKGROUND: Increasing evidence suggests that postoperative infection is associated with poorer long-term outcome in various malignancies. However, the mechanism of poor prognosis induced by postoperative infection has not been clearly explained. We sought to determine whether abdominal infection promotes cancer metastases in a murine liver metastasis model, and to investigate the role of liver natural killer (NK) cells on antitumor immunity during abdominal infection. METHODS: Female BALB/c (8-10 weeks old) mice were inoculated with NL-17 colon cancer cells into the spleen and then subjected to abdominal infection induced by cecal ligation and puncture (CLP) or sham treatment. The extent of liver metastases and cytokine production in the serum and liver were investigated. Cell fraction and cytotoxic activities of liver mononuclear cells (MNCs) were elucidated. RESULTS: CLP mice had poorer survival and their serum levels of IL-6, -10, and -12p70 were significantly elevated on day 1 compared with sham-treated and control mice. No obvious differences in cytokine levels of the liver homogenates were identified among the three groups, except IL-12p70 levels in CLP mice on day 7 significantly decreased. The cytotoxic activities of liver MNCs were significantly suppressed in CLP mice soon after tumor inoculation. Flow cytometry revealed a decrease in NK cells in the liver and perforin and granzyme B expression levels. CONCLUSIONS: Abdominal infection promoted liver metastases in a murine liver metastasis model, which may be partially caused by a decrease in the number and activity of NK cells during abdominal infection.

Basal expression of insulin-like growth factor 1 receptor determines intrinsic resistance of cancer cells to a phosphatidylinositol 3-kinase inhibitor ZSTK474.

Drug resistance often critically limits the efficacy of molecular targeted drugs. Although pharmacological inhibition of phosphatidylinositol 3-kinase (PI3K) is an attractive therapeutic strategy for cancer therapy, molecular determinants for efficacy of PI3K inhibitors (PI3Kis) remain unclear. We previously identified that overexpression of insulin-like growth factor 1 receptor (IGF1R) contributed to the development of drug resistance after long-term exposure to PI3Kis. In this study, we examined the involvement of basal IGF1R expression in intrinsic resistance of drug-naïve cancer cells to PI3Kis and whether inhibition of IGF1R overcomes the resistance. We found that cancer cells highly expressing IGF1R showed resistance to dephosphorylation of Akt and subsequent antitumor effect by ZSTK474 treatment. Knockdown of IGF1R by siRNAs facilitated the dephosphorylation and enhanced the drug efficacy. These cells expressed tyrosine-phosphorylated insulin receptor substrate 1 at high levels, which was dependent on basal IGF1R expression. In these cells, the efficacy of ZSTK474 in vitro and in vivo was improved by its combination with the IGF1R inhibitor OSI-906. Finally, we found a significant correlation between the basal expression level of IGF1R and the inefficacy of ZSTK474 in an in vivo human cancer panel, as well as in vitro. These results suggest that basal IGF1R expression affects intrinsic resistance of cancer cells to ZSTK474, and IGF1R is a promising target to improve the therapeutic efficacy. The current results provide evidence of combination therapy of PI3Kis with IGF1R inhibitors for treating IGF1R-positive human cancers.

Comprehensive transcriptomic analysis of molecularly targeted drugs in cancer for target pathway evaluation.

Targeted therapy is a rational and promising strategy for the treatment of advanced cancer. For the development of clinical agents targeting oncogenic signaling pathways, it is important to define the specificity of compounds to the target molecular pathway. Genome-wide transcriptomic analysis is an unbiased approach to evaluate the compound mode of action, but it is still unknown whether the analysis could be widely applicable to classify molecularly targeted anticancer agents. We comprehensively obtained and analyzed 129 transcriptomic datasets of cancer cells treated with 83 anticancer drugs or related agents, covering most clinically used, molecularly targeted drugs alongside promising inhibitors of molecular cancer targets. Hierarchical clustering and principal component analysis revealed that compounds targeting similar target molecules or pathways were clustered together. These results confirmed that the gene signatures of these drugs reflected their modes of action. Of note, inhibitors of oncogenic kinase pathways formed a large unique cluster, showing that these agents affect a shared molecular pathway distinct from classical antitumor agents and other classes of agents. The gene signature analysis further classified kinome-targeting agents depending on their target signaling pathways, and we identified target pathway-selective signature gene sets. The gene expression analysis was also valuable in uncovering unexpected target pathways of some anticancer agents. These results indicate that comprehensive transcriptomic analysis with our database (http://scads.jfcr.or.jp/db/cs/) is a powerful strategy to validate and re-evaluate the target pathways of anticancer compounds.

Identification of a molecular target of kurahyne, an apoptosis-inducing lipopeptide from marine cyanobacterial assemblages.

In 2014, we isolated kurahyne, an acetylene-containing lipopeptide, from a marine cyanobacterial assemblage of Lyngbya sp. Kurahyne exhibited growth-inhibitory activity against human cancer cells, and induced apoptosis in HeLa cells. However, its mode of action is not yet clear. To elucidate its mode of action, we carried out several cell-based assays, and identified the intracellular target molecule of kurahyne as sarco/endoplasmic reticulum Ca(2+) ATPase (SERCA). In addition, we found that kurahyne inhibited the differentiation of macrophages into osteoclasts.

Synthesis of dansyl-labeled probe of thiophene analogue of annonaceous acetogenins for visualization of cell distribution and growth inhibitory activity toward human cancer cell lines.

The convergent synthesis of the dansyl-labeled probe of the thiophene-3-carboxamide analogue of annonaceous acetogenins, which shows potent antitumor activity, was accomplished by two asymmetric alkynylations of the 2,5-diformyl THF equivalent with an alkyne having a thiophene moiety and another alkyne tagged with a dansyl group. The growth inhibitory profiles toward 39 human cancer cell lines revealed that the probe retained the biological function of its mother compound, and would be useful for studying cellular activity.

Ridaifen G, tamoxifen analog, is a potent anticancer drug working through a combinatorial association with multiple cellular factors.

Ridaifen-G (RID-G), a tamoxifen analog that we previously synthesized, has potent growth inhibitory activity against various cancer cell lines. Tamoxifen is an anticancer drug known to act on an estrogen receptor (ER) and other proteins. However, our previous studies interestingly suggested that the mechanism of action of RID-G was different from that of tamoxifen. In order to investigate the molecular mode of action of RID-G, we developed a novel chemical genetic approach that combined a phage display screen with a statistical analysis of drug potency and gene expression profiles in thirty-nine cancer cell lines. Application of this method to RID-G revealed that three proteins, calmodulin (CaM), heterogeneous nuclear ribonucleoproteins A2/B1 (hnRNP A2/B1), and zinc finger protein 638 (ZNF638) were the candidates of direct targets of RID-G. Moreover, cell lines susceptible to RID-G show similar expression profiles of RID-G target genes. These results suggest that RID-G involves CaM, hnRNP A2/B1, and ZNF638 in its growth inhibitory activity.

Extensive analysis of signaling pathway molecules in breast cancer: association with clinicopathological characteristics.

BACKGROUND: The aim of this study was to extensively analyze the signaling pathway molecules in breast cancer and to explore candidate biomarkers for clinicopathological relevance. METHODS: We assessed the expression of key factors in cell signaling, namely p-AKT, cyclin D1, P27, p-p70S6 K, p-4EBP1, and p-MAPK/ERK, within 338 invasive breast cancer patients. These factors were immunohistochemically examined in tumor tissues and assessed by staining score. Staining scores were analyzed by a clustering method to devise a new classification based on pathway activity. We investigated the relationships among staining scores, the clustering classification, and patient characteristics. RESULTS: The proportion of patients displaying high expression levels were as follows: p-AKT, 75%; cyclin D1, 12%; P27, 53%; p-p70S6 K, 37%; p-4EBP1, 19%; and p-MAPK/ERK, 3%. Patients were classified into two groups on the basis of staining scores. Group 1 (39%) included more positive cases for p-4EBP1, p-MAPK/ERK, and p-p70S6 K and fewer positive cases for P27 and cyclin D1 than Group 2 (61%). The clustering classification was significantly related to subgrouping by hormone receptor and HER2 (P < 0.001), nuclear grade (P < 0.001) and histological subtype (P = 0.034). A strong positive correlation was identified between p-AKT and P27, cyclin D1 and P27, p-p70S6 K and p-4EBP1, p-p70S6 K and p-MAPK/ERK, and between p-4EBP1 and p-MAPK/ERK. Levels of p-p70S6 K were significantly related to recurrence in both univariate (RR = 0.75, P < 0.001) and multivariate (RR = 0.71, P = 0.049) analyses. CONCLUSIONS: The present study helps us to understand the characteristics of signaling pathway status in breast cancers. Moreover, p-p70S6 K expression may be of use in predicting clinical outcome.

Antitumor effects of tyropeptin-boronic acid derivatives: New proteasome inhibitors.

The proteasome degrades numerous regulatory proteins that are critical for tumor growth. Thus, proteasome inhibitors are promising antitumor agents. New proteasome inhibitors, such as tyropeptins and tyropeptin-boronic acid derivatives, have a potent inhibitory activity. Here we report the antitumor effects of two new tyropeptin-boronic acid derivatives, AS-06 and AS-29. AS-06 and AS-29 significantly suppress the degradation of the proteasome-sensitive fluorescent proteins in HEK293PS cells, and induce the accumulation of ubiquitinated proteins in human multiple myeloma cells. We show that these derivatives also suppress the degradation of the NF-κB inhibitor IκB-α and the nuclear translocation of NF-κB p65 in multiple myeloma cells, resulting in the inhibition of NF-κB activation. Furthermore, we demonstrate that AS-06 and AS-29 induce apoptosis through the caspase-8 and caspase-9 cascades. In a xenograft mouse model, i.v. administration of tyropeptin-boronic acid derivatives inhibits proteasome in tumors and clearly suppresses tumor growth in mice bearing human multiple myeloma. Our results indicate that tyropeptin-boronic acid derivatives could be lead therapeutic agents against human multiple myeloma.

In vitro multifaceted activities of a specific group of novel phosphatidylinositol 3-kinase inhibitors on hotspot mutant PIK3CA.

BACKGROUND: As accumulating evidences suggest close involvement of phosphatidylinositol 3-kinase (PI3K) in cancer, novel PI3K inhibitors such as ZSTK474, GDC-0941, NVP-BEZ235 and BKM-120 have been developed for cancer therapy. A high frequency of hotspot mutations known as E542K, E545K and H1047R in the PIK3CA gene, which encodes the catalytic subunit of PI3Kα, has been found in various types of human cancers. The hotspot PIK3CA mutations also lead to resistance to therapeutics targeting epidermal growth factor receptor (EGFR), further suggesting that inhibition of hotspot mutant PIK3CA be required for a PI3K inhibitor as anticancer drug candidate. METHODS: To investigate the activity of the novel PI3K inhibitors on the hotspot mutant PIK3CA, we determined the inhibition against the respective recombinant mutant PI3Kαs by biochemical assay. We further examined the activity at cellular background by determining the effect on phosphorylation of Akt (Ser473), and that on the growth of cancer cells. In addition, apoptosis and autophagy in cells with or without hotspot PIK3CA mutation induced by the four inhibitors were investigated. RESULTS: Our results indicated that each inhibitor exhibit comparable activity on the hotspot mutant PI3Kα to that on the wild type, which was further demonstrated by the cell-based assays. No clear correlation was shown between the PIK3CA genetic status and the sensitivity for apoptosis or autophagy induction. Interestingly, among the 4 PI3K inhibitors, BKM-120 is the weakest in PI3K inhibitory potency, but induces most potent apoptosis, suggesting that BKM-120 might have a unique mode of action. CONCLUSIONS: Our result shows that the PI3K inhibitors exhibit potent activity on both hotspot mutant and wild type PI3Kα, suggesting they might be used to treat patients with or without PIK3CA mutation when approved.

In vitro antitumor activity of stellettin B, a triterpene from marine sponge Jaspis stellifera, on human glioblastoma cancer SF295 cells.

Stellettin B was isolated from marine sponge Jaspis stellifera. In vitro antitumor activities were investigated on 39 human cancer cell lines. Stellettin B exhibited highly potent inhibition against the growth of a human glioblastoma cell line SF295, with a GI50 of 0.01 µM. In contrast, stellettin B showed very weak inhibitory activity on normal cell lines including HMEC, RPTEC, NHBE and PrEC, with GI50s higher than 10 µM, suggesting its relatively selective cytotoxicity against human cancer cells compared to normal human cell lines. We then focused on the antitumor activity of this compound on SF295 cells. Flow cytometric analysis indicated that stellettin B induced apoptosis in SF295 cells in a concentration-dependent manner. Further study indicated that stellettin B increased the production of ROS, the activity of caspase 3/7, as well as the cleavage of PARP, each of which is known to be involved in apoptosis. To investigate the molecular mechanism for cell proliferation inhibition and apoptosis induction, effect on the phosphorylation of several signal proteins of PI3K/Akt and RAS/MAPK pathways was examined. Stellettin B inhibited the phosphorylation of Akt potently, with no activity on p-ERK and p-p38, suggesting that inhibition of PI3K/Akt pathway might be involved in the antiproliferative and apoptosis-inducing effect. However, homogenous time-resolved fluorescence (HTRF) assay indicated that stellettin B did not inhibit PI3K activity, suggesting that the direct target might be signal protein upstream of Akt pathway other than PI3K.

Identification of transporters associated with Etoposide sensitivity of stomach cancer cell lines and methotrexate sensitivity of breast cancer cell lines by quantitative targeted absolute proteomics.

Membrane transporter proteins may influence the sensitivity of cancer cells to anticancer drugs that can be recognized as substrates. The purpose of this study was to identify proteins that play a key role in the drug sensitivity of stomach and breast cancer cell lines by measuring the absolute protein expression levels of multiple transporters and other membrane proteins and examining their correlation to drug sensitivity. Absolute protein expression levels of 90 membrane proteins were examined by quantitative targeted absolute proteomics using liquid chromatography-linked tandem mass spectrometry. Among them, 11 and 14 membrane proteins, including transporters, were present in quantifiable amounts in membrane fraction of stomach cancer and breast cancer cell lines, respectively. In stomach cancer cell lines, the protein expression level of multidrug resistance-associated protein 1 (MRP1) was inversely correlated with etoposide sensitivity. MK571, an MRP inhibitor, increased both the cell-to-medium ratio of etoposide and the etoposide sensitivity of MRP1-expressing stomach cancer cell lines. In breast cancer cell lines, the protein expression level of reduced folate carrier 1 (RFC1) was directly correlated with methotrexate (MTX) sensitivity. Initial uptake rate and steady-state cell-to-medium ratio of [(3)H]MTX were correlated with both RFC1 expression level and MTX sensitivity. These results suggest that MRP1 modulates the etoposide sensitivity of stomach cancer cell lines and RFC1 modulates the MTX sensitivity of breast cancer cell lines. Our results indicate that absolute quantification of multiple membrane proteins could be a useful strategy for identification of candidate proteins involved in drug sensitivity.

AMF-26, a novel inhibitor of the Golgi system, targeting ADP-ribosylation factor 1 (Arf1) with potential for cancer therapy.

ADP-ribosylation factor 1 (Arf1) plays a major role in mediating vesicular transport. Brefeldin A (BFA), a known inhibitor of the Arf1-guanine nucleotide exchange factor (GEF) interaction, is highly cytotoxic. Therefore, interaction of Arf1 with ArfGEF is an attractive target for cancer treatment. However, BFA and its derivatives have not progressed beyond the pre-clinical stage of drug development because of their poor bioavailability. Here, we aimed to identify novel inhibitors of the Arf1-ArfGEF interaction that display potent antitumor activity in vivo but with a chemical structure distinct from that of BFA. We exploited a panel of 39 cell lines (termed JFCR39) coupled with a drug sensitivity data base and COMPARE algorithm, resulting in the identification of a possible novel Arf1-ArfGEF inhibitor AMF-26, which differed structurally from BFA. By using a pulldown assay with GGA3-conjugated beads, we demonstrated that AMF-26 inhibited Arf1 activation. Subsequently, AMF-26 induced Golgi disruption, apoptosis, and cell growth inhibition. Computer modeling/molecular dynamics (MD) simulation suggested that AMF-26 bound to the contact surface of the Arf1-Sec7 domain where BFA bound. AMF-26 affected membrane traffic, including the cis-Golgi and trans-Golgi networks, and the endosomal systems. Furthermore, using AMF-26 and its derivatives, we demonstrated that there was a significant correlation between cell growth inhibition and Golgi disruption. In addition, orally administrated AMF-26 (83 mg/kg of body weight; 5 days) induced complete regression of human breast cancer BSY-1 xenografts in vivo, suggesting that AMF-26 is a novel anticancer drug candidate that inhibits the Golgi system, targeting Arf1 activation.

Development of a gene expression database and related analysis programs for evaluation of anticancer compounds.

Genome-wide transcriptional expression analysis is a powerful strategy for characterizing the biological activity of anticancer compounds. It is often instructive to identify gene sets involved in the activity of a given drug compound for comparison with different compounds. Currently, however, there is no comprehensive gene expression database and related application system that is; (i) specialized in anticancer agents; (ii) easy to use; and (iii) open to the public. To develop a public gene expression database of antitumor agents, we first examined gene expression profiles in human cancer cells after exposure to 35 compounds including 25 clinically used anticancer agents. Gene signatures were extracted that were classified as upregulated or downregulated after exposure to the drug. Hierarchical clustering showed that drugs with similar mechanisms of action, such as genotoxic drugs, were clustered. Connectivity map analysis further revealed that our gene signature data reflected modes of action of the respective agents. Together with the database, we developed analysis programs that calculate scores for ranking changes in gene expression and for searching statistically significant pathways from the Kyoto Encyclopedia of Genes and Genomes database in order to analyze the datasets more easily. Our database and the analysis programs are available online at our website (http://scads.jfcr.or.jp/db/cs/). Using these systems, we successfully showed that proteasome inhibitors are selectively classified as endoplasmic reticulum stress inducers and induce atypical endoplasmic reticulum stress. Thus, our public access database and related analysis programs constitute a set of efficient tools to evaluate the mode of action of novel compounds and identify promising anticancer lead compounds.

Novel tamoxifen derivative Ridaifen-B induces Bcl-2 independent autophagy without estrogen receptor involvement.

Autophagy is a self-proteolysis process in eukaryotic cells that results in the sequestering of intracellular proteins and organelles in autophagosomes. Activation of autophagy progress continued growth of some tumors, instead extensive autophagy induces cell death. In a previous study, we synthesized a novel tamoxifen derivative, Ridaifen (RID)-B. RID-B induced mitochondria-involved apoptosis even in estrogen receptor (ER)-negative cells. Since tamoxifen induces autophagy other than apoptosis, we treated ER-negative Jurkat cells with RID-B in the present study. RID-B treatment induced apoptosis and LC3 and lysosome colocalization, which results in the formation of autolysosomes. Western blotting revealed that LC3 was converted to LC3-I to LC3-II with RID-B treatment, suggesting that RID-B induced autophagy without ER involvement. Moreover, overexpression of the anti-apoptotic protein Bcl-2 suppressed the RID-B-induced cell death, but not the induction of autophagy. These results presumed that RID-B-induced autophagy is independent of Bcl-2, making RID-B-induced autophagy different from RID-B-induced apoptosis. Since Beclin 1 level is unchanged during RID-B treatment, RID-B induced autophagy pathway is Bcl-2/Beclin1 independent noncanonical pathway.

Search for novel anti-tumor agents from ridaifens using JFCR39, a panel of human cancer cell lines.

To overcome the heterogeneous nature of cancer, the search for potent anti-cancer drug candidates with new modes of action is essential. For that purpose, we prepared forty-eight Ridaifens (RIDs), a novel series of tamoxifen-derivatives. Then, we screened them, searching for novel candidates for a new class of anti-cancer drug using a panel of human cancer cell lines (JFCR39) and by a binding assay to estrogen receptor α (ERα). First, the growth inhibition of the forty-eight RIDs against JFCR39 was evaluated. Forty RIDs showed higher growth-inhibitory activity than that of tamoxifen. The structure-activity relationship (SAR) study revealed that the aminoalkoxyphenyl groups at the C-1 position and the common central ethylenic bond were important in retaining a high level of growth-inhibitory activity. Subsequently, the ERα binding activity of all the RIDs was measured by a competitive binding assay. The SAR study for ERα binding activity indicated that both the phenyl group and the ethyl group at the C-2 position in the ethylenic bond were essential. Based on the screenings, we identified RID-SB1 and RID-SB8, which demonstrated potent tumor growth inhibition but had completely lost ERα binding activity. Furthermore, the COMPARE analysis using JFCR39 suggested that RID-SB1 and RID-SB8 had different molecular modes of action compared to those of the current anti-cancer drugs including tamoxifen. These results indicate that RID-SB1 and RID-SB8 are interesting candidates for novel anti-cancer agents with unique modes of action.