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Background: Histone deacetylase (HDAC) inhibitors have been reported to exhibit immunomodulatory activities, including the upregulation of major histocompatibility complex class I (MHC class I). Although the immunoproteasome plays a pivotal role in MHC class I antigen presentation, its effect on immunotherapy for clear cell renal cell carcinoma (ccRCC) remains unclear., Methods: This study assessed whether OBP-801, a novel HDAC inhibitor, affects the expression of immunoproteasome subunits and subsequently the MHC class-I-mediated anti-cancer immunity in ccRCC. We analyzed the data of 531 patients with ccRCC from the Cancer Genome Atlas Kidney Clear Cell Carcinoma database. We further evaluated the treatment efficacy of the combination of OBP-801 and anti-PD-1 in a ccRCC mouse model., Results: Low molecular mass polypeptide (LMP) 2 was correlated most positively with CD3E, CD8A, and CD8B expression and estimated CD8 + T cell number. In vitro studies showed that OBP-801 upregulated MHC class I presentation by inducing LMP2 expression in the ccRCC cell lines RENCA, 786-O, and Caki-1. In vivo studies in a syngeneic mouse model with subcutaneous implantation of RENCA cells showed that OBP-801 treatment increased the percentage of CD45 + CD3e + T cells in tumor-infiltrating lymphocytes. The combination of anti-PD-1 antibody and OBP-801 enhanced the anti-tumor effect, LMP2 protein expression, and MHC class I presentation in tumor cells. MHC class I presentation in the tumors of each mouse was positively correlated with the percentage of CD45 + CD3e + T cells., Conclusions: Our results demonstrate that OBP-801 promotes MHC class I presentation through LMP2 upregulation in tumor cells and thereby potentiates PD-1-targeting therapy. These data suggest that the combination of OBP-801 and anti-PD-1 treatment is a promising therapeutic strategy for ccRCC.

Lazertinib, a novel third-generation epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI), demonstrates marked efficacy in EGFR-mutant lung cancer. However, resistance commonly develops, prompting consideration of therapeutic strategies to overcome initial drug resistance mechanisms. This study aimed to elucidate the adaptive resistance to lazertinib and advocate novel combination treatments that demonstrate efficacy in preventing resistance as a first-line treatment for EGFR mutation-positive NSCLC. We found that AXL knockdown significantly inhibited lung cancer cell viability in the presence of lazertinib, indicating that AXL activation contributes to lazertinib resistance. However, long-term culture with a combination of lazertinib and AXL inhibitors led to residual cell proliferation and increased the MCL-1 expression level, which was mediated by the nuclear translocation of the transcription factor YAP. Triple therapy with an MCL-1 or YAP inhibitor in combination with lazertinib and an AXL inhibitor significantly reduced cell viability and increased the apoptosis rate. These results demonstrate that AXL and YAP/MCL-1 signals contribute to adaptive lazertinib resistance in EGFR-mutant lung cancer cells, suggesting that the initial dual inhibition of AXL and YAP/MCL-1 might be a highly effective strategy in eliminating lazertinib-resistant cells., (© 2024 The Author(s). Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Background: Recent therapeutic strategies for KRAS-mutated cancers that inhibit the MAPK pathway have attracted considerable attention. The RAF/MEK clamp avutometinib (VS-6766/CH5126766/RO5126766/CKI27) is promising for patients with KRAS-mutated cancers. Although avutometinib monotherapy has shown clinical activity in patients with KRAS-mutated cancers, effective combination strategies will be important to develop., Methods: Using a phosphorylation kinase array kit, we explored the feedback mechanism of avutometinib in KRAS-mutated NSCLC cells, and investigated the efficacy of combining avutometinib with inhibitors of the feedback signal using in vitro and in vivo experiments. Moreover, we searched for a biomarker for the efficacy of combination therapy through an in vitro study and analysis using the The Cancer Genome Atlas Programme dataset., Results: Focal adhesion kinase (FAK) phosphorylation/activation was increased after avutometinib treatment and synergy between avutometinib and FAK inhibitor, defactinib, was observed in KRAS-mutated NSCLC cells with an epithelial rather than mesenchymal phenotype. Combination therapy with avutometinib and defactinib induced apoptosis with upregulation of Bim in cancer cells with an epithelial phenotype in an in vitro and in vivo study., Conclusions: These results demonstrate that the epithelial-mesenchymal transition status may be a promising biomarker for the efficacy of combination therapy with avutometinib and defactinib in KRAS-mutated NSCLC., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Few treatment options exist for pleural mesothelioma (PM), which is a progressive malignant tumor. However, the efficacy of molecular-targeted monotherapy is limited, and further therapeutic strategies are warranted to treat PM. Recently, the cancer cell-cycle checkpoint inhibitors have attracted attention because they disrupt cell-cycle regulation. Here, we aimed to establish a novel combinational therapeutic strategy to inhibit the cell-cycle checkpoint kinase, ATR in PM cells. The siRNA screening assay showed that anexelekto (AXL) knockdown enhanced cell growth inhibition when exposed to ATR inhibitors, demonstrating the synergistic effects of the ATR and AXL combination in some PM cells. The AXL and ATR inhibitor combination increased cell apoptosis via the Bim protein and suppressed cell migration when compared with each monotherapy. The combined therapeutic targeting of AXL and ATR significantly delayed regrowth compared with monotherapy. Thus, optimal AXL and ATR inhibition may potentially improve the PM outcome., (©2023 The Authors; Published by the American Association for Cancer Research.)

γ-Glutamylcyclotransferase (GGCT) is highly expressed in multiple types of cancer tissues and its knockdown suppresses the growth of cancer cells in vitro and in vivo. Although GGCT is a promising target for cancer therapy, the mechanisms underlying the antitumor effects remain unclear. The knockdown of GGCT inhibited the MEK-ERK pathway, and activated the tumor suppressor retinoblastoma gene (RB) at the protein level in cancer cell lines. c-Met was down-regulated by the knockdown of GGCT in cancer cells and its overexpression attenuated the dephosphorylation of RB and cell cycle arrest induced by the knockdown of GGCT in lung cancer A549 cells. STAT3 is a transcription factor that induces c-Met expression. STAT3 phosphorylation and its nuclear expression level were decreased in GGCT-depleted A549 and prostate cancer PC3 cells. The simultaneous knockdown of AMPK and GGCT restored the down-regulated expression of c-Met, and attenuated the dephosphorylation of STAT3 and MEK-ERK-RB induced by the knockdown of GGCT in PC3 cells. An intraperitoneal injection of a GGCT inhibitor decreased c-Met protein expression in a mouse xenograft model of PC3 cells. These results suggest that the knockdown of GGCT activates the RB protein by inhibiting the STAT3-c-Met-MEK-ERK pathway via AMPK activation., (© 2023. The Author(s).)

Anaplastic lymphoma kinase (ALK)-tyrosine kinase inhibitors rarely elicit complete responses in patients with advanced ALK-rearranged non-small cell lung cancer (NSCLC), as a small population of tumor cells survives due to adaptive resistance. Therefore, we focused on the mechanisms underlying adaptive resistance to lorlatinib and therapeutic strategies required to overcome them. We found that epidermal growth factor receptor (EGFR) signaling was involved in the adaptive resistance to lorlatinib in ALK-rearranged NSCLC, activation of which was induced by heparin-binding EGF-like growth factor production via c-Jun activation. EGFR inhibition halted ALK-rearranged lung cancer cell proliferation by enhancing ALK inhibition-induced apoptosis via suppression of Bcl-xL. Xenograft models showed that the combination of EGFR inhibitor and lorlatinib considerably suppressed tumor regrowth following cessation of these treatments. This study provides new insights regarding tumor evolution due to EGFR signaling after lorlatinib treatment and the development of combined therapeutic strategies for ALK-rearranged lung cancer., (© 2023. The Author(s).)

Background: Recent advances have been achieved in the genetic diagnosis and therapies against malignancies due to a better understanding of the molecular mechanisms underlying carcinogenesis. Since active preventive methods are currently insufficient, the further development of appropriate preventive strategies is desired., Methods: We searched for drinks that reactivate the functions of tumor-suppressor retinoblastoma gene (RB) products and exert anti-inflammatory and antioxidant effects. We also examined whether lactic acid bacteria increased the production of the cancer-specific anti-tumor cytokine, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), in human, and examined whether the RB-reactivating drinks with lactic acid bacteria decreased azoxymethane-induced rat colon aberrant crypt foci (ACF) and aberrant crypts (ACs) in vivo., Results: Kakadu plum juice and pomegranate juice reactivated RB functions, which inhibited the growth of human colon cancer LIM1215 cells by G1 phase arrest. These juices also exerted anti-inflammatory and antioxidant effects. Lactiplantibacillus (L.) pentosus S-PT84 was administered to human volunteers and increased the production of TRAIL. In an in vivo study, Kakadu plum juice with or without pomegranate juice and S-PT84 significantly decreased azoxymethane-induced rat colon ACF and ACs., Conclusions: RB is one of the most important molecules suppressing carcinogenesis, and to the best of our knowledge, this is the first study to demonstrate that natural drinks reactivated the functions of RB. As expected, Kakadu plum juice and pomegranate juice suppressed the growth of LIM1215 cells by reactivating the functions of RB, and Kakadu plum juice with or without pomegranate juice and S-PT84 inhibited rat colon ACF and ACs. Therefore, this mixed juice has potential as a novel candidate for cancer prevention.

Anticancer drug delivery by small molecules offers a number of advantages over conventional macromolecular drug delivery systems. We previously developed phenylcyclopropylamine (PCPA)-drug conjugates (PDCs) as small-molecule-based drug delivery vehicles for targeting lysine-specific demethylase 1 (LSD1)-overexpressing cancers. In this study, we applied this PDC strategy to the HDAC-inhibitory anticancer agent vorinostat. Among three synthesized PCPA or arylcyclopropylamine (ACPA)-vorinostat conjugates 1 , 9 , and 32 , conjugate 32 with a 4-oxybenzyl linker showed sufficient stability in buffer solutions, potent LSD1 inhibition, efficient LSD1-dependent vorinostat release, and potent and selective antiproliferative activity toward LSD1-expressing human breast cancer and small-cell lung cancer cell lines. These results indicate that the conjugate selectively releases vorinostat in cancer cells. A similar strategy may be applicable to other anticancer drugs., Competing Interests: The authors declare no competing financial interest., (© 2022 American Chemical Society.)

Aspirin has gained great attention as a cancer preventive agent. Our previous study revealed that the low-dose aspirin prevents colorectal tumor recurrence in Japanese patients with colorectal adenomas and/or adenocarcinomas, whereas aspirin increases risks in smokers and has no effects on regular drinkers. Our recent study revealed that aspirin reduces polyp growth in Japanese patients with familial adenomatous polyposis (FAP). In this study, we have studied the association of genotypes of alcohol metabolizing enzymes (ADH1B and ALDH2) on aspirin's efficacy of suppressing polyp growth (≥5 mm) in a total of 81 Japanese patients with FAP. Our study revealed that aspirin showed significant preventive effects for patients with ADH1B -AA and AA+GA types [OR = 0.21; 95% confidence interval (CI), 0.05-0.95, and OR = 0.31; 95% CI, 0.10-0.95, respectively], and for patients with ALDH2 -GG and GG+GA types (OR = 0.10; 95% CI, 0.01-0.92, and OR = 0.29; 95% CI, 0.09-0.94, respectively), but not for patients with ADH1B -GG and GA+GG types, and ALDH2 -AA and GA+AA types. In addition, substantial preventive effects of aspirin were seen for patients with ADH1B -AA type who do not drink regularly (<3 times/week, OR = 0.11; 95% CI, 0.02-0.78), where a statistically significant interaction between aspirin and ADH1B was observed ( P interaction = 0.036). Results from this exploratory study strongly indicate that aspirin is beneficial in prevention of polyp growth for patients with FAP with ADH1B -AA and AA+GA types, and ALDH2 -GG and GG+GA types. Taken together, we propose ADH1B and ALDH2 as candidate markers for the personalized prevention by aspirin., Significance: Aspirin is beneficial to patients with FAP with ADH1B -AA and AA+GA types or ALDH2 -GG and GG+GA types. ADH1B and ALDH2 genotypes can be the markers for the personalized prevention of colorectal cancer by aspirin., Competing Interests: K. Mure, H. Ishikawa, M. Mutoh, K. Wakabayashi, T. Sakai, and S. Tanaka report a grant from Japan Agency for Medical Research and Development during the conduct of the study. Y. Takeuchi reports personal fees from Olympus, Boston Scientific Japan, Daiichi-Sankyo, Miyarisan Pharmaceutical, Asuka Pharmacoceutical, AstraZeneca, EA Pharma, Zeria Pharmaceutical, Fujifilm, Kaneka Medical, and Kyorin Pharmaceutical outside the submitted work. No other disclosures were reported., (© 2022 The Authors; Published by the American Association for Cancer Research.)

Edible plant-derived nanovesicles have been explored as effective materials for preventing colorectal cancer (CRC) incidence, dependent on gene status, as a K-Ras-activating mutation via the macropinocytosis pathway. Approximately 70% of CRC harbors the p53 mutation, which is strongly associated with a poor prognosis for CRC. However, it has not been revealed whether p53 inactivation activates the macropinocytosis pathway or not. In this study, we investigated parental cells, wild-type or null for p53 treated with Citrus limon L. -derived nanovesicles, as potential materials for CRC prevention. Using ultracentrifugation, we obtained C. limon L .-derived nanovesicles, the diameters of which were approximately 100 nm, similar to that of the exosomes derived from mammalian cells. C. limon L .-derived nanovesicles showed inhibitory effects on cell growth in not p53-wild, but also in p53-inactivated CRC cells. Furthermore, we revealed that the macropinocytosis pathway is activated by p53 inactivation and C. limon L. -derived nanovesicles were up taken via the macropinocytosis pathway. Notably, although C. limon L .-derived nanovesicles contained citrate, the inhibitory effects of citrate were not dependent on the p53 status. We thus provide a novel mechanism for the growth inhibition of C. limon L .-derived nanovesicles via macropinocytosis and expect to develop a functional food product containing them for preventing p53-inactivation CRC incidence.

Multiple myeloma (MM) is characterized by remarkable cytogenetic/molecular heterogeneity among patients and intraclonal diversity even in a single patient. We previously demonstrated that PDPK1, the master kinase of series of AGC kinases, is universally active in MM, and plays pivotal roles in cell proliferation and cell survival of myeloma cells regardless of the profiles of cytogenetic and genetic abnormalities. This study investigated the therapeutic efficacy and mechanism of action of dual blockade of two major PDPK1 substrates, RSK2 and AKT, in MM. The combinatory treatment of BI-D1870, an inhibitor for N-terminal kinase domain (NTKD) of RSK2, and ipatasertib, an inhibitor for AKT, showed the additive to synergistic anti-tumor effect on human MM-derived cell lines (HMCLs) with active RSK2-NTKD and AKT, by enhancing apoptotic induction with BIM and BID activation. Moreover, the dual blockade of RSK2 and AKT exerted robust molecular effects on critical gene sets associated with myeloma pathophysiologies, such as those with MYC, mTOR, STK33, ribosomal biogenesis, or cell-extrinsic stimuli of soluble factors, in HMCLs. These results provide the biological and molecular rationales for the dual-targeting strategy for RSK2 and AKT, which may overcome the therapeutic difficulty due to cytogenetic/molecular heterogeneity in MM.

As colon cancer is one of the most common cancers in the world, practical prevention strategies for colon cancer are needed. Recently, treatment with aspirin and/or 5-aminosalicylic acid-related agents was reported to reduce the number of intestinal polyps in patients with familial adenomatous polyposis. To evaluate the mechanism of aspirin and 5-aminosalicylic acid for suppressing the colon polyp growth, single and combined effects of 5-aminosalicylic acid and sodium salicylate (metabolite of aspirin) were tested in the two human colon cancer cells with different cyclooxygenase-2 expression levels and intestinal polyp-derived cells from familial adenomatous polyposis model mouse. The combination induced cell-cycle arrest at the G1 phase along with inhibition of cell growth and colony-forming ability in these cells. The combination reduced cyclin D1 via proteasomal degradation and activated retinoblastoma protein. The combination inhibited the colony-forming ability of mouse colonic mucosa cells by about 50% and the colony-forming ability of mouse intestinal polyp-derived cells by about 90%. The expression level of cyclin D1 in colon mucosa cells was lower than that in intestinal polyp-derived cells. These results suggest that this combination may be more effective in inhibiting cell growth of intestinal polyps through cyclin D1 down-regulation., Competing Interests: No potential conflicts of interest were disclosed., (Copyright © 2022 JCBN.)

EGFR-T790M mutation is a major mechanism underlying acquired resistance to first- and second-generation EGFR tyrosine kinase inhibitors (EGFR-TKIs) in lung cancer with mutated EGFR. However, differences in the biological characteristics of T790M tumors based on treatment regimens with each generation of EGFR-TKI are not fully understood. We established cell lines with acquired resistance harboring EGFR-T790M mutation derived from xenograft tumors treated with each generation of EGFR-TKI and examined their biological characteristics with respect to third-generation EGFR-TKI osimertinib sensitivity. Second-generation EGFR-TKI dacomitinib-resistant cells with T790M-exhibited higher sensitivity to osimertinib than first-generation EGFR-TKI gefitinib-resistant cells with T790M via inhibition of AKT and ERK signaling and promotion of apoptosis. Furthermore, gefitinib-resistant cells showed enhanced intratumor heterogeneity accompanied by genomic instability and activation of alternative resistance mechanisms compared with dacomitinib-resistant cells; this suggests that the maintenance of EGFR dependency after acquiring resistance might depend on the type of EGFR-TKI. Our results demonstrate that the progression of tumor heterogeneity via both genetic and non-genetic mechanisms might affect osimertinib sensitivity in tumors with acquired resistance harboring EGFR-T790M mutation., (© 2022 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

Anaplastic lymphoma kinase-tyrosine kinase inhibitors (ALK-TKIs) have shown dramatic efficacy in patients with ALK-rearranged lung cancer; however, complete response in these patients is rare. Here, we investigated the molecular mechanisms underlying the emergence and maintenance of drug-tolerant cells in ALK-rearranged lung cancer. Cell based-assays demonstrated that HER3 activation and mesenchymal-to-epithelial transition, mediated through ZEB1 proteins, help maintain cell survival and induce the emergence of ALK-TKI-tolerant cells. Compared with ALK-TKIs alone, cotreatment with pan-HER inhibitor afatinib and ALK-TKIs prevented tumor regrowth, leading to the eradication of tumors in ALK-rearranged tumors with mesenchymal features. Moreover, pre-treatment vimentin expression in clinical specimens obtained from patients with ALK-rearranged lung cancer was associated with poor ALK-TKI treatment outcomes. These results demonstrated that HER3 activation plays a pivotal role in the emergence of ALK-TKI-tolerant cells. Furthermore, the inhibition of HER3 signals combined with ALK-TKIs dramatically improves treatment outcomes for ALK-rearranged lung cancer with mesenchymal features., (© 2022. The Author(s).)

Various molecular-targeting drugs have markedly improved the treatment of patients with breast cancer. As yet, therapies for triple-negative breast cancer are mainly cytotoxic agents. To investigate the novel therapy for triple-negative breast cancer, we herein examined the effects of a new combination therapy comprising a RAF/MEK inhibitor CH5126766, also known as VS-6766, which we originally discovered, and eribulin. The combination of CH5126766 and eribulin potently inhibited cell growth in the triple-negative breast cancer cell lines tested. The underlying mechanism in the efficacy of this combination treatment in vitro and in vivo was due to enhanced apoptosis through the suppression of survivin and Bcl-2 family proteins. We also showed the suppressed expression of programmed cell death ligand 1 (PD-L1) in combination therapy in vivo. We found that combination therapy with eribulin and CH5126766 for triple-negative breast cancer inhibited cell growth by apoptosis and raised a possibility that immune responses through suppression of PD-L1 might partially contribute to inhibition of tumor growth, indicating the potential of this combination as a novel strategy for triple-negative breast cancer., (© 2021 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Cigarette smoking and alcohol consumption are major risk factors for lifestyle-related diseases. Although it has been reported that the combination of these habits worsens risks, the underlying mechanism remains elusive. Reactive carbonyl species (RCS) cause chemical modifications of biological molecules, leading to alterations in cellular signaling pathways, and total RCS levels have been used as a lipid peroxidation marker linked to lifestyle-related diseases. In this study, at least 41 types of RCS were identified in the lipophilic fraction of plasma samples from 40 subjects using liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS). Higher levels of 10 alkanals, 5 trans -2-alkenals, 1 cis -4-alkenal, and 3 alkadienals were detected in the smoking/drinking group ( N = 10) as compared to those with either habit ( N = 10 each) or without both habits ( N = 10) in the analysis of covariances adjusted for age and BMI. The levels of 3 alkanals, 1 trans -2-alkenal, 1 alkadienal, and 1 4-hydroxy-2-alkenal in the smoking/drinking group were significantly higher than those in the no-smoking/drinking and no-smoking/no-drinking groups. These results strongly indicate that the combination of cigarette smoking and alcohol drinking synergistically increases the level and variety of RCS in the circulating blood, and may further jeopardize cellular function.

Purpose: To evaluate the presence of ocular surface mucin in patients with atopic and vernal keratoconjunctivitis (AKC/VKC), we investigated the mRNA expression levels of SAM-pointed domain-containing ETS-like factor ( SPDEF ) and mucin-related genes on the ocular surface., Methods: Nineteen patients with AKC or VKC were divided into two groups based on the severity of the disease as determined by their clinical scores for AKC/VKC: the stable group and the active group. Impression cytology was performed in all patients using filter paper, and the expression levels of SPDEF , MUC1 , MUC4 , MUC5AC , MUC16 , and eotaxin-2 mRNA were determined by real-time reverse-transcription polymerase chain reaction., Results: The results showed that the expression levels of SPDEF and MUC5AC mRNA in the active group were significantly decreased compared with those in the stable group. Furthermore, clinical scores were significantly negatively correlated with the expression levels of SPDEF mRNA and significantly positively correlated with the expression levels of eotaxin-2, which is a biomarker for eosinophilic inflammation on the ocular surface. Cluster analysis classified the patients with AKC/VKC into three clusters, and the stable group was divided into two clusters according to the condition of ocular surface mucin., Conclusions: Ocular surface mucin in patients with AKC/VKC is altered in accordance with the clinical severity of the disease., Competing Interests: J. S. has previously received honoraria from Santen Pharmaceutical Co., Ltd., Senju Pharmaceutical Co., Ltd., and Alcon Pharmaceuticals, outside the submitted work. S. Y., N. I., Y. T., A. T., and M. H. declare that they have no conflicts of interest., (Copyright © 2021 Mariko Horinaka et al.)

Purpose: Currently, an optimal therapeutic strategy comprising molecularly targeted agents for treating EGFR -mutated non-small cell lung cancer (NSCLC) patients with acquired resistance to osimertinib is not available. Therefore, the initial therapeutic intervention is crucial for the prolonged survival of these patients. The activation of anexelekto (AXL) signaling is known to be associated with intrinsic and acquired resistance to EGFR tyrosine kinase inhibitors (EGFR-TKIs). In this study, we investigated the best therapeutic strategy to combat AXL-induced tolerance to EGFR-TKIs using the novel AXL inhibitor ONO-7475., Experimental Design: We examined the efficacy of ONO-7475 in combination with EGFR-TKIs in EGFR -mutated NSCLC cells using in vitro and in vivo experiments. We investigated the correlation between AXL expression in tumors and clinical outcomes with osimertinib for EGFR -mutated NSCLC patients with acquired resistance to initial EGFR-TKIs., Results: ONO-7475 sensitized AXL-overexpressing EGFR -mutant NSCLC cells to the EGFR-TKIs osimertinib and dacomitinib. In addition, ONO-7475 suppressed the emergence and maintenance of EGFR-TKI-tolerant cells. In the cell line-derived xenograft models of AXL-overexpressing EGFR -mutated lung cancer treated with osimertinib, initial combination therapy of ONO-7475 and osimertinib markedly regressed tumors and delayed tumor regrowth compared with osimertinib alone or the combination after acquired resistance to osimertinib. AXL expression in EGFR-TKI refractory tumors did not correlate with the sensitivity of osimertinib., Conclusions: These results demonstrate that ONO-7475 suppresses the emergence and maintenance of tolerant cells to the initial EGFR-TKIs, osimertinib or dacomitinib, in AXL-overexpressing EGFR -mutated NSCLC cells, suggesting that ONO-7475 and osimertinib is a highly potent combination for initial treatment., (©2020 American Association for Cancer Research.)

Purpose: Myxofibrosarcoma is characterized by a high rate of recurrence after surgery. Since myxofibrosarcoma is refractory to conventional cytotoxic chemotherapy, the established radical treatment is primary wide resection. The effects of histone deacetylase (HDAC) inhibitors on myxofibrosarcoma have not yet been investigated. Therefore, the main purpose of the present study was to examine the effects of a HDAC inhibitor on myxofibrosarcoma., Methods: The effects of the HDAC inhibitor OBP-801 on human myxofibrosarcoma cells were examined using cell viability assay, flow cytometric analysis of the cell cycle and apoptosis, and Western blotting. The effects of combinations of OBP-801 with pazopanib or Akt-mTOR inhibitors were also investigated using cell viability assay., Results: OBP-801 inhibited the growth of myxofibrosarcoma NMFH-1 and NMFH-2 cells. It also induced cell cycle arrest at the G2 phase and apoptosis in both cell lines. The inhibitory effects of pazopanib and Akt-mTOR inhibitors on the growth of myxofibrosarcoma cells were enhanced by the combination with OBP-801., Conclusions: The present results demonstrated that OBP-801 exerted therapeutic effects in myxofibrosarcoma in both single and concomitant administrations. Therefore, OBP-801 has potential as a novel treatment for myxofibrosarcoma.

Sulforaphane and Lactobacilli induce apoptosis in several cancer cells. Sulforaphane, a dietary isothiocyanate, is an attractive agent due to its potent anticancer effects. Sulforaphane suppresses the proliferation of various cancer cells in vitro and in vivo . The present study investigated the effect of sulforaphane and a co-culture with Lactobacillus -treated peripheral blood mononuclear cells (PBMCs) in human colon cancer cells. The combination markedly induced apoptosis in human colon cancer HCT116 and SW480 cells. A pan-caspase inhibitor markedly inhibited apoptosis, and a tumor necrosis factor (TNF) receptor/Fc chimera partially inhibited apoptosis in both cells. The amount of TNFα secretion in the culture supernatant was significantly increased by co-culture with Lactobacillus -treated normal human PBMCs. On the other hand, the expression of cellular inhibitor of apoptosis-2 (cIAP-2), an anti-apoptotic protein, was increased by co-culture with Lactobacillus -treated PBMCs in colon cancer cells, but sulforaphane treatment significantly suppressed the induction of cIAP-2. The present results revealed that sulforaphane enhances apoptosis in human colon cancer cells under co-culture with Lactobacillus -treated PBMCs via the TNFα signaling pathway., (Copyright © 2019, Spandidos Publications.)

The prognosis of muscle-invasive bladder cancer with metastasis is poor. There have been no therapeutic improvements for many years, and an innovative therapy for muscle-invasive bladder cancer has been awaited to replace the conventional cytotoxic chemotherapy. Here, we show a candidate method for the treatment of bladder cancer. The combined treatment with a novel histone deacetylase (HDAC) inhibitor, OBP-801, and celecoxib synergistically inhibited cell growth and markedly induced apoptosis through the caspase-dependent pathway in high-grade bladder cancer cells. Furthermore, the combined treatment induced expression of death receptor 5 (DR5). We identified that knockdown of DR5 by small interfering RNA (siRNA) significantly suppressed apoptosis by the combined treatment. Therefore, we conjectured that the apoptosis induced by OBP-801 and celecoxib is at least partially dependent on DR5. However, it was interesting that the combined treatment drastically suppressed expression of DR5 ligand, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). These data suggest that there is no involvement of TRAIL in the induction of apoptosis by the combination, regardless of the dependence of DR5. Moreover, xenograft studies using human bladder cancer cells showed that the combined therapy suppressed tumor growth by upregulating expressions of DR5 and Bim. The inhibition of tumor growth was significantly more potent than that of each agent alone, without significant weight loss. This combination therapy provided a greater benefit than monotherapy in vitro and in vivo These data show that the combination therapy with OBP-801 and celecoxib is a potential novel therapeutic strategy for patients with muscle-invasive bladder cancer. Mol Cancer Ther; 15(9); 2066-75. ©2016 AACR., (©2016 American Association for Cancer Research.)

Although many chemotherapeutic strategies against cancer have been developed, pancreatic cancer is one of the most aggressive and intractable types of malignancies. Therefore, new strategies and anti-cancer agents are necessary to treat this disease. Metformin is a widely used drug for type-2 diabetes, and is also known as a promising candidate anti-cancer agent from recent studies in vitro and in vivo. However, the mechanisms of metformin's anti-cancer effects have not been elucidated. We demonstrated that metformin suppressed the expression of miR-221, one of the most well-known oncogenic microRNAs, in human pancreatic cancer PANC-1 cells. Moreover, we showed that the down-regulation of miR-221 by metformin caused G1-phase arrest via the up-regulation of p27, one of the direct targets of miR-221. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is also a promising agent for cancer treatment. While recent studies showed that treatment with only TRAIL was not effective against pancreatic cancer cells, the present data showed that metformin sensitized p53-mutated pancreatic cancer cells to TRAIL. Metformin induced the expressions of death receptor 5 (DR5), a receptor for TRAIL, and Bim with a pro-apoptotic function in the downstream of TRAIL-DR5 pathway. We suggest that the up-regulation of these proteins may contribute to sensitization of TRAIL-induced apoptosis. The combination therapy of metformin and TRAIL could therefore be effective in the treatment of pancreatic cancer.

Although melanoma is the most aggressive skin cancer, recent advances in BRAF and/or MEK inhibitors against BRAF-mutated melanoma have improved survival rates. Despite these advances, a treatment strategy targeting NRAS-mutated melanoma has not yet been elucidated. We discovered CH5126766/RO5126766 as a potent and selective dual RAF/MEK inhibitor currently under early clinical trials. We examined the activity of CH5126766/RO5126766 in a panel of malignant tumor cell lines including melanoma with a BRAF or NRAS mutation. Eight cell lines including melanoma were assessed for their sensitivity to the BRAF, MEK, or RAF/MEK inhibitor using in vitro growth assays. CH5126766/RO5126766 induced G1 cell cycle arrest in two melanoma cell lines with the BRAF V600E or NRAS mutation. In these cells, the G1 cell cycle arrest was accompanied by up-regulation of the cyclin-dependent kinase inhibitor p27 and down-regulation of cyclinD1. CH5126766/RO5126766 was more effective at reducing colony formation than a MEK inhibitor in NRAS- or KRAS-mutated cells. In the RAS-mutated cells, CH5126766/RO5126766 suppressed the MEK reactivation caused by a MEK inhibitor. In addition, CH5126766/RO5126766 suppressed the tumor growth in SK-MEL-2 xenograft model. The present study indicates that CH5126766/RO5126766 is an attractive RAF/MEK inhibitor in RAS-mutated malignant tumor cells including melanoma.

A combined therapy of sulindac sulfide and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising strategy for the treatment of cancer. Sulindac sulfide had been shown to induce the expression of death receptor 5 (DR5), a receptor for TRAIL, and sensitize cancer cells to TRAIL-induced apoptosis; however, the molecular mechanism underlying the upregulation of DR5 has not yet been elucidated. We demonstrate here that myeloid zinc finger 1 (MZF1) mediates the induction of DR5 by sulindac sulfide. Sulindac sulfide induced the expression of DR5 at the protein and mRNA levels in colon cancer SW480 cells. Furthermore, sulindac sulfide increased DR5 promoter activity. We showed that sulindac sulfide stimulated DR5 promoter activity via the -301 to -253 region. This region contained a putative MZF1-binding site. Site-directed mutations in the site abrogated the enhancement in DR5 promoter activity by sulindac sulfide. MZF1 directly bound to the putative MZF1-binding site of the DR5 promoter and the binding was increased by sulindac sulfide. The expression of MZF1 was also increased by sulindac sulfide, and MZF1 siRNA attenuated the upregulation of DR5 by sulindac sulfide. These results indicate that sulindac sulfide induces the expression of DR5 by up-regulating MZF1.

Methylglyoxal is an essential component in glycolysis and is known to be an inducer of apoptosis. Glyoxalase I (GLO1) metabolizes and inactivates methylglyoxal. GLO1 is known to be overexpressed in cancer cells and causes resistance to anticancer agents. We show for the first time that methylglyoxal treatment or the silencing of GLO1 enhances sensitivity to the promising anticancer agent TRAIL in malignant tumor cells. Methylglyoxal suppressed the expression of antiapoptotic factors, X-linked inhibitor of apoptosis protein (XIAP), survivin, cIAP1, Bcl-2, and Bcl-xL, without affecting TRAIL receptors, DR4 and DR5. Knockdown of XIAP or survivin by siRNA also enhanced TRAIL-induced apoptosis, indicating that downregulation of XIAP and survivin expression by methylglyoxal contributes to the enhancement of TRAIL activity. Furthermore, methylglyoxal decreased NF-κB activity with or without TRAIL treatment. On the other hand, the knockdown of GLO1 by siRNA enhanced TRAIL-induced apoptosis via the downregulation of XIAP and survivin expression. In conclusion, our results strongly suggest that sensitivity to TRAIL is increased by inhibition of the glyoxalase pathway and that the combination of TRAIL with methylglyoxal or glyoxalase inhibitors may be useful for a novel combination chemotherapy., (©2012 AACR.)

Anthracycline drugs are potent anti-tumor agents. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a death ligand with promising anti-cancer effects. However, some tumor types develop resistance to TRAIL. We examined the effect of aclarubicin (ACR), an anthracycline, in combination with TRAIL. The combination of TRAIL and ACR synergistically induced apoptosis in human acute lymphoblastic leukemia Jurkat cells and human lung cancer A549 cells. In contrast, another anthracycline, doxorubicin (DOX), only slightly sensitized Jurkat cells and A549 cells to TRAIL-induced apoptosis, with weaker enhancement of death receptor 5 (DR5) expression than ACR. The RNase protection assay, real time RT-PCR and western blot demonstrated that ACR upregulated the expression of a TRAIL receptor, DR5. Caspase inhibitors and dominant negative DR5 efficiently reduced the apoptotic response to the treatment with ACR and TRAIL, indicating that the combined effect depends on caspase activities and the interaction between TRAIL and its receptor. ACR but not DOX increased the activity of the DR5 gene promoter in Jurkat cells carrying a mutation in the p53 gene, suggesting that ACR upregulates DR5 expression through p53-independent transcription. These results suggest the combination of TRAIL and ACR to be a promising treatment for malignant tumors., (© 2011 Japanese Cancer Association.)

Malignant tumors carry a high risk of death, and the prevention of malignant tumors is a crucial issue in preventive medicine. To this end, many chemopreventive agents have been tested, but the effects of single agents have been found to be insufficient to justify clinical trials. We have therefore hypothesized that combinations of different chemopreventive agents may synergistically enhance the preventive effect of chemopreventive agents used singly. To provide the treating physician with some guideline by which to choose the most effective agents to be combined, we propose a strategy which we have termed the "combination-oriented molecular-targeting prevention" of cancer. As the molecular target of our model, we focused on tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), which specifically causes apoptosis in malignant tumor cells. Many of these agents were found to up-regulate the expression of death receptor 5, a TRAIL receptor. They were also found to synergistically induce apoptosis in malignant tumor cells when combined with TRAIL. Here, we strongly advocate that the strategy of "combination-oriented molecular-targeting prevention" of cancer will be a practical approach for chemoprevention against human malignant tumors.

Purpose: The clinically relevant histone deacetylase inhibitors (HDI) valproic acid (VPA) and suberoylanilide hydroxamic acid exert variable antitumor activities but increase therapeutic efficacy when combined with other agents. The natural endogenous ligand of peroxisome proliferator-activated receptor gamma 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)) is a potent antineoplastic agent. Therefore, we investigated whether these HDIs in combination with 15d-PGJ(2) could show synergistic antitumor activity in colon cancer DLD-1 cells., Experimental Design: Cell viability was determined using a Cell Counting Kit-8 assay. Apoptosis and reactive oxygen species (ROS) generation were determined using flow cytometry analysis. Western blotting and real-time reverse transcription-PCR analysis were carried out to investigate the expression of apoptosis-related molecules. Mice bearing DLD-1 xenograft were divided into four groups (n = 5) and injected everyday (i.p.) with diluent, VPA (100 mg/kg), 15d-PGJ(2) (5 mg/kg), or a combination for 25 days., Results: HDI/15d-PGJ(2) cotreatments synergistically induced cell death through caspase-dependent apoptosis in DLD-1 cells. Moreover, HDIs/15d-PGJ(2) caused histone deacetylase inhibition, leading to subsequent ROS generation and endoplasmic reticulum stress to decrease the expression of antiapoptotic molecules Bcl-X(L) and XIAP and to increase that of proapoptotic molecules CAAT/enhancer binding protein homologous protein and death receptor 5. Additionally, VPA/15d-PGJ(2) cotreatment induced ROS-dependent apoptosis in other malignant tumor cells and was more effective than a VPA or 15d-PGJ(2) monotherapy in vivo., Conclusions: Cotreatments with the clinically relevant HDIs and the endogenous peroxisome proliferator-activated receptor gamma ligand 15d-PGJ(2) are promising for the treatment of a broad spectrum of malignant tumors.

Methotrexate (MTX) has been used to treat various hematological malignancies. Since MTX prevents tumor cells from proliferating by inhibiting dihydrofolate reductase (DHFR), DHFR expression is a key determinant of resistance to MTX in malignant hematological tumor cells. The antiproliferative effect of MTX was significantly enhanced by the knockdown of DHFR expression by siRNA in Jurkat cells. Therefore, a novel strategy down-regulating DHFR expression seems promising for enhancing sensitivity to MTX. We found that SU9516, a cyclin-dependent kinase inhibitor, reduced the expression of both DHFR mRNA and protein. Moreover, we found that DHFR promoter activity was attenuated by SU9516 dependent on the E2F site. Finally, pretreatment with SU9516 significantly enhanced sensitivity to MTX in a colony formation assay. We conclude that a combination of cyclin-dependent kinase inhibitors and MTX may be useful for overcoming resistance to MTX.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is an endogenous cytokine that induces apoptosis in malignant tumor cells. Here, we show for the first time that lactobacilli induce TRAIL production in human peripheral blood mononuclear cells (PBMC). Treatment with lactobacilli induced TRAIL on the cell surface of PBMC and in culture medium. The TRAIL production induced by lactobacilli partially depends on IFN-alpha and IFN-gamma. Lactobacilli treatment facilitated NK activity of PBMC against prostate cancer cells. Moreover, TRAIL neutralization antibody efficiently prevented the NK activity. Our results indicate that lactobacilli facilitate NK activity through TRAIL production, and raise the possibility of a new TRAIL-based strategy against malignant tumors., (2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Allopurinol has been used for the treatment of gout and conditions associated with hyperuricemia for several decades. We explored the potential of allopurinol on cancer treatment. Allopurinol did not expose cytotoxicity as a single treatment in human hormone refractory prostate cancer cell lines, PC-3 and DU145. However, allopurinol drastically induced apoptosis of PC-3 and DU145 in combination with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), which is a promising candidate for anticancer agent but its efficacy is limited by the existence of resistant cancer cells. We examined the underlying mechanism by which allopurinol overcomes the resistance of prostate cancer cells to TRAIL. Allopurinol up-regulated the expression of a proapoptotic TRAIL receptor, death receptor 5 (DR5). Allopurinol increased DR5 protein, mRNA, and promoter activity. Using DR5 small interfering RNA (siRNA), we showed that allopurinol-mediated DR5 up-regulation contributed to the enhancement of TRAIL effect by allopurinol. Furthermore, we examined the mechanism of allopurinol-mediated DR5 up-regulation. DR5 promoter activity induced by allopurinol was diminished by a mutation of a CAAT/enhancer binding protein homologous protein (CHOP)-binding site. In addition, allopurinol also increased CHOP expression, suggesting that allopurinol induced DR5 expression via CHOP. Allopurinol possesses the activity of a xanthine oxidase (XO) inhibitor. We used XO siRNA instead of allopurinol. XO siRNA also up-regulated DR5 and CHOP expression and sensitized the prostate cancer cells to TRAIL-induced apoptosis. Here, we show the novel potential of allopurinol in cancer treatment and indicate that the combination of allopurinol with TRAIL is effective strategy to expand the TRAIL-mediated cancer therapy.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is one of the most promising candidates for new cancer therapeutics. A current problem is that some cancers still remain resistant to TRAIL. We show for the first time that a naturally occurring flavonoid, baicalein, overcomes TRAIL resistance in cancer cells. The combination of baicalein and TRAIL effectively induced apoptosis in TRAIL-resistant colon cancer SW480 cells. Baicalein up-regulated the expression of death receptor 5 (DR5) among TRAIL receptors at the mRNA and protein levels. Suppression of this up-regulation with small interfering RNA (siRNA) efficiently reduced the apoptosis induced by TRAIL and baicalein, suggesting that the sensitization was mediated through DR5 induction. Moreover, baicalein also overcame TRAIL resistance with DR5 up-regulation in prostate cancer PC3 cells. Of note, the combination of TRAIL and baicalein hardly induced apoptosis in normal human cells, such as blood cells and hepatocytes. Baicalein increased DR5 promoter activity, and this enhanced activity was diminished by mutation of a CCAAT/enhancer-binding protein homologous protein (CHOP)-binding site in SW480 cells. In SW480 cells, CHOP siRNA blocked both functions of baicalein. CHOP expression was induced by baicalein in SW480 cells; however, in PC3 cells, baicalein scarcely induced CHOP and mutation of the CHOP-binding site did not abrogate the DR5 promoter activation by baicalein. Interestingly, baicalein induced reactive oxygen species (ROS) and a ROS scavenger prevented DR5 expression and TRAIL sensitization in PC3 but not SW480 cells. These results indicate that, using two different pathways, baicalein exposes cancer surveillance of TRAIL and overcomes TRAIL resistance in cancer cells.

Objectives: Isoliquiritigenin is a chalcone derivative with potential in cancer chemoprevention. Although tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anti-cancer agent, some cancer cells are resistant to TRAIL treatment. Current studies have tried to overcome TRAIL-resistant cancer cells. Here, we show for the first time that isoliquiritigenin overcomes TRAIL resistance in colon cancer HT29 cells., Methods: HT29 cells were treated with isoliquiritigenin and/or TRAIL, and apoptosis induction was detected by flow cytometry and fluorescence microscopy. Protein expression relating to the TRAIL pathway was analyzed by Western blotting., Results: A single treatment with isoliquiritigenin scarcely induced apoptosis in HT29 cells. Combined treatment with suboptimal concentrations of isoliquiritigenin and TRAIL markedly induced apoptosis, however. The effect was blocked by a pan-caspase inhibitor and a caspase-3, 8, 9, or 10 inhibitor, suggesting that the combination facilitates caspase-dependent apoptosis. Furthermore, the apoptosis induced by isoliquiritigenin and TRAIL was blocked by a dominant negative form of the TRAIL receptor. This result indicates that the combined effect is caused by specific interaction between TRAIL and its receptors. Isoliquiritigenin increased the amount of DR5 protein among TRAIL receptors. Isoliquiritigenin did not significantly increase levels of the Bcl-2 family proteins Bcl-2, Bcl-xL, and BAX., Conclusions: Our results suggest that isoliquiritigenin has the potential to overcome resistance to TRAIL in cancer cells and its chemopreventive effects may depend on TRAIL function.

Lipoxygenases induce malignant tumor progression and lipoxygenase inhibitors have been considered as promising anti-tumor agents. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is one of the most promising candidates for new cancer therapeutics. Combined treatment with nordihydroguaiaretic acid (NDGA), a lipoxygenase inhibitor, and TRAIL markedly induced apoptosis in Jurkat T-cell leukemia cells at suboptimal concentrations for each agent. The combined treatment efficiently activated caspase-3, -8 and -10, and Bid. The underling mechanism by which NDGA enhanced TRAIL-induced apoptosis was examined. NDGA did not change the expression levels of anti-apoptotic factors, Bcl-x(L), Bcl-2, cIAP-1, XIAP and survivin. The expression of death receptor-related genes was investigated and it was found that NDGA specifically up-regulated the expression of death receptor 5 (DR5) at mRNA and protein levels. Down-regulation of DR5 by small interfering RNA prevented the sensitizing effect of NDGA on TRAIL-induced apoptosis. Furthermore, NDGA sensitized prostate cancer and colorectal cancer cells to TRAIL-induced apoptosis. In contrast, NDGA neither enhanced TRAIL-induced apoptosis nor up-regulated DR5 expression in normal peripheral blood mononuclear cells. Another lipoxygenase inhibitor, AA861, also up-regulated DR5 and sensitized Jurkat and DU145 cells to TRAIL. These results indicate that lipoxygenase inhibitors augment the apoptotic efficiency of TRAIL through DR5 up-regulation in malignant tumor cells, and raise the possibility that the combination of lipoxygenase inhibitor and TRAIL is a promising strategy for malignant tumor treatment.

Carotenoids are compounds contained in foods and possess anticarcinogenic activity. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising candidate for cancer therapeutics due to its ability to induce apoptosis selectively in cancer cells. However, some tumors remain tolerant to TRAIL-induced apoptosis. Therefore, it is important to develop agents that overcome this resistance. We show, for the first time, that certain carotenoids sensitize cancer cells to TRAIL-induced apoptosis. Combined treatment with halocynthiaxanthin, a dietary carotenoid contained in oysters and sea squirts, and TRAIL drastically induced apoptosis in colon cancer DLD-1 cells, whereas each agent alone only slightly induced apoptosis. The combination induced nuclear condensation and poly(ADP-ribose) polymerase cleavage, which are major features of apoptosis. Various caspase inhibitors could attenuate the apoptosis induced by this combination. Furthermore, the dominant-negative form of a TRAIL receptor could block the apoptosis, suggesting that halocynthiaxanthin specifically facilitated the TRAIL signaling pathway. To examine the molecular mechanism of the synergistic effect of the combined treatment, we did an RNase protection assay. Halocynthiaxanthin markedly up-regulated a TRAIL receptor, death receptor 5 (DR5), among the death receptor-related genes, suggesting a possible mechanism for the combined effects. Moreover, we examined whether other carotenoids also possess the same effects. Peridinin, but not alloxanthin, diadinochrome, and pyrrhoxanthin, induced DR5 expression and sensitized DLD-1 cells to TRAIL-induced apoptosis. These results indicate that the combination of certain carotenoids and TRAIL is a new strategy to overcome TRAIL resistance in cancer cells.

Sulforaphane (SFN), a naturally occurring isothiocyanate, is an attractive agent because of its potent anticancer effects. SFN suppresses the proliferation of various cancer cells in vitro and in vivo. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is also one of the most promising candidates for cancer therapeutics owing to its ability to selectively induce apoptosis in tumor cells. In this study, we report that SFN enhances TRAIL-induced apoptosis in human osteosarcoma cells, Saos2 and MG63. The apoptosis induced by co-treatment with SFN and TRAIL was markedly blocked by a dominant negative form of the TRAIL receptor or caspase inhibitors. The combined use of SFN and TRAIL effectively induced Bid cleavage and the activation of caspases 8, 10, 9 and 3 at ineffective concentrations for each agent. SFN upregulated the expression of death receptor 5 (DR5), a receptor for TRAIL, at mRNA and protein levels in a dose-dependent manner. In addition, the SFN-mediated sensitization to TRAIL was reduced by DR5 siRNA, suggesting that the sensitization was at least partially mediated through the induction of DR5 expression. Furthermore, SFN sensitized TRAIL-induced apoptosis in a p53-independent manner. On the other hand, SFN neither induced DR5 protein expression or enhanced TRAIL-induced apoptosis in normal human peripheral blood mononuclear cells. Thus, combined treatment with SFN and TRAIL might be a promising therapy for osteosarcoma.

A collagen-degrading thermophile, Geobacillus collagenovorans MO-1, extracellularly produces a collagenolytic protease with a large molecular mass. Complete nucleotide sequencing of this gene after gene cloning revealed that the collagenolytic protease is a member of the subtilisin family of serine proteases and consists of a signal sequence for secretion, a prosequence for maturation, a catalytic region, 14 direct repeats of 20 amino acids at the C terminus, and a region with unknown function intervening between the catalytic region and the numerous repeats. Since the unusual repeats are most likely to be cleaved in the secreted form of the enzyme, the intervening region was investigated to determine whether it participates in collagen binding to facilitate collagen degradation. It was found that the mature collagenolytic protease containing the intervening region at the C terminus bound collagen but not the other insoluble proteins, elastin and keratin. Furthermore, the intervening region fused with glutathione S-transferase showed a collagen-binding ability comparable to that of the mature collagenolytic protease. The collagen-binding ability was finally attributed to two-thirds of the intervening region which is rich in beta-strands and is approximately 35 kDa in molecular mass. In the collagenolytic protease from strain MO-1, hydrogen bonds most likely predominate over the hydrophobic interaction for collagen binding, since a higher concentration of NaCl released collagen from the enzyme surface but a nonionic detergent could not. To the best of our knowledge, this is the first report of a thermophilic collagenolytic protease containing the collagen-binding segment.

15-Deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)), the terminal derivative of the PGJ series, is emerging as a potent antineoplastic agent among cyclopentenone prostaglandins derivatives and also known as the endogenous ligand of peroxisome proliferator-activated receptor gamma (PPARgamma). On the other hand, death receptor 5 (DR5) is a specific receptor for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), which is one of the most promising candidates for new cancer therapeutics. Here, we report that 15d-PGJ(2) induces DR5 expression at both mRNA and protein levels, resulting in the synergistic sensitization of TRAIL-induced apoptosis in human neoplastic cells, such as Jurkat human leukemia cells or PC3 human prostate cancer cells. 15d-PGJ(2) significantly increased DR5 mRNA stability, whereas it did not activate DR5 promoter activity. Synthetic PPARgamma agonists, such as pioglitazone or rosiglitazone, did not mimic the DR5-inducing effects of 15d-PGJ(2), and a potent PPARgamma inhibitor GW9662 failed to block DR5 induction by 15d-PGJ(2), suggesting PPARgamma-independent mechanisms. Cotreatment with 15d-PGJ(2) and TRAIL enhanced the sequential activation of caspase-8, caspase-10, caspase-9, caspase-3, and Bid. DR5/Fc chimera protein, zVAD-fmk pancaspase inhibitor, and caspase-8 inhibitor efficiently blocked the activation of these apoptotic signal mediators and the induction of apoptotic cell death enhanced by cotreatment with 15d-PGJ(2) and TRAIL. Moreover, a double-stranded small interfering RNA targeting DR5 gene, which suppressed DR5 up-regulation by 15d-PGJ(2), significantly attenuated apoptosis induced by cotreatment with 15d-PGJ(2) and TRAIL. These results suggest that 15d-PGJ(2) is a potent sensitizer of TRAIL-mediated cancer therapeutics through DR5 up-regulation.

Dietary flavonoid apigenin is expected to have preventive and therapeutic potential against malignant tumors. In this report, we show for the first time that apigenin markedly induces the expression of death receptor 5 (DR5) and synergistically acts with exogenous soluble recombinant human tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) to induce apoptosis in malignant tumor cells. TRAIL is a promising candidate for cancer therapeutics due to its ability to selectively induce apoptosis in cancer cells. The combined use of apigenin and TRAIL at suboptimal concentrations induces Bcl-2-interacting domain cleavage and the activation of caspases-8, -10, -9, and -3. Furthermore, human recombinant DR5/Fc chimera protein and caspase inhibitors dramatically inhibit apoptosis induced by the combination of apigenin and TRAIL. On the other hand, apigenin-mediated induction of DR5 expression is not observed in normal human peripheral blood mononuclear cells. Moreover, apigenin does not sensitize normal human peripheral blood mononuclear cells to TRAIL-induced apoptosis. These results suggest that this combined treatment with apigenin and TRAIL might be promising as a new therapy against malignant tumors.

Luteolin, a naturally occurring flavonoid, induces apoptosis in various cancer cells. Little is known however concerning the underlying molecular mechanisms responsible for this activity. In this report, we reveal a novel mechanism by which luteolin-induced apoptosis occurs, and show for the first time that the apoptosis by luteolin is mediated through death receptor 5 (DR5) upregulation. Luteolin markedly induced the expression of DR5, along with Bcl-2-interacting domain cleavage and the activation of caspase-8, -10, -9 and -3. In addition, suppression of DR5 expression with siRNA efficiently reduced luteolin-induced caspase activation and apoptosis. Human recombinant DR5/Fc also inhibited luteolin-induced apoptosis. On the other hand, luteolin induced neither DR5 protein expression nor apoptosis in normal human peripheral blood mononuclear cells. These results suggest that DR5 induced by luteolin plays a role in luteolin-induced apoptosis, and raises the possibility that treatment with luteolin might be promising as a new therapy against cancer., (Oncogene (2005) 24, 7180-7189. doi:10.1038/sj.onc.1208874; published online 11 July 2005.)

Death receptor 5 (DR5/TRAIL-R2) is an apoptosis-inducing membrane receptor for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo2L). In this study, we showed that tunicamycin, a naturally occurring antibiotic, is a potent enhancer of TRAIL-induced apoptosis through up-regulation of DR5 expression. Tunicamycin significantly sensitized PC-3, androgen-independent human prostate cancer cells, to TRAIL-induced apoptosis. The tunicamycin-mediated enhancement of TRAIL-induced apoptosis was markedly blocked by a recombinant human DR5/Fc chimeric protein. Tunicamycin and TRAIL cooperatively activated caspase-8, -10, -9, and -3 and Bid cleavage and this activation was also blocked in the presence of the DR5/Fc chimera. Tunicamycin up-regulated DR5 expression at the mRNA and protein levels in a dose-dependent manner. Furthermore, the tunicamycin-mediated sensitization to TRAIL was efficiently reduced by DR5 small interfering RNA, suggesting that the sensitization was mediated through induction of DR5 expression. Tunicamycin increased DR5 promoter activity and this enhanced activity was diminished by mutation of a CHOP-binding site. In addition, suppression of CHOP expression by small interfering RNA reduced the tunicamycin-mediated induction of DR5. Of note, tunicamycin-mediated induction of CHOP and DR5 protein expression was not observed in normal human peripheral blood mononuclear cells. Moreover, tunicamycin did not sensitize the cells to TRAIL-induced apoptosis. Thus, combined treatment with tunicamycin and TRAIL may be a promising candidate for prostate cancer therapy.

Combined treatment with a proteasome inhibitor and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising strategy for cancer therapy. Proteasome inhibitors induce the expression of death receptor 5 (DR5), a receptor for TRAIL, and sensitize cancer cells to TRAIL-induced apoptosis; however, the molecular mechanism of DR5 up-regulation has not been elucidated. In this study, we report that CCAAT/enhancer-binding protein homologous protein (CHOP) is a regulator of DR5 induction by proteasome inhibitor MG132. MG132 induced DR5 expression at a protein and mRNA level in prostate cancer DU145 cells. Furthermore, MG132 increased DR5 promoter activity. Using a series of deletion mutant plasmids containing DR5 promoters of various sizes, we found that MG132 stimulated the promoter activity via the region of -289 to -253. This region contained a CHOP-binding site. Site-directed mutation of the site abrogated the promoter activity enhanced by MG132. An electrophoretic mobility shift assay showed that CHOP directly bound to the MG132-responsive site on the DR5 promoter. Expression of the CHOP protein was increased with MG132 along with DR5 up-regulation. Furthermore, CHOP small interfering RNA attenuated the DR5 up-regulation due to MG132. These results indicate that the proteasome inhibitor MG132 induces DR5 expression through CHOP up-regulation.

Genistein is the most abundant isoflavone of soybeans and has been shown to cause growth arrest in various human cancer cell lines. However, the precise mechanism for this is still unclear. We report here that the growth arrest and DNA damage-inducible gene 45 (gadd45) gene is induced by genistein via its promoter in a DU145 human prostate cancer cell line. The binding of transcription factor nuclear factor-Y to the CCAAT site of the gadd45 promoter appears to be important for this activation by genistein.

Death receptor 5 (DR5) is a receptor for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). TRAIL is a promising candidate for cancer therapeutics due to its ability to induce apoptosis selectively in cancer cells. Here, we report that histone deacetylase inhibitors (HDACIs) such as trichostatin A (TSA), sodium butyrate, and suberoylanilide hydroxamic acid (SAHA) upregulated DR5 expression in various human malignant tumor cells. An RNase protection assay demonstrated that HDACIs induced DR5 mRNA markedly but not that of other death receptor family members in Jurkat cells. HDACIs increased DR5 mRNA and protein in a dose- and time-dependent manner. We also show TSA increased DR5 promoter activity using a luciferase promoter assay. Furthermore, we demonstrated that HDACIs strongly sensitized exogenous soluble recombinant human TRAIL-induced apoptosis synergistically in Jurkat and HL-60 cells that were tolerant to TRAIL alone. The combined use of HDACIs and TRAIL in suboptimal concentrations induced Bid cleavage and activation of caspase-8, -10, -3, and -9. Human recombinant DR5/Fc chimera protein, zVAD-fmk pancaspase inhibitor, and caspase-8 and -10 inhibitors efficiently reduced apoptosis induced by cotreatment with HDACIs and TRAIL. Furthermore, TSA did not significantly induce DR5 protein and HDACIs did not enhance TRAIL-induced apoptosis in normal human peripheral blood mononuclear cells. These results suggest that this combined treatment with HDACIs and TRAIL is a promising strategy for new cancer therapeutics.