Novel drug-resistance mechanisms of pemetrexed-treated non-small cell lung cancer.

Pemetrexed (PEM) improves the overall survival of patients with advanced non-small cell lung cancer (NSCLC) when administered as maintenance therapy. However, PEM resistance often appears during the therapy. Although thymidylate synthase is known to be responsible for PEM resistance, no other mechanisms have been investigated in detail. In this study, we explored new drug resistance mechanisms of PEM-treated NSCLC using two combinations of parental and PEM-resistant NSCLC cell lines from PC-9 and A549. PEM increased the apoptosis cells in parental PC-9 and the senescent cells in parental A549. However, such changes were not observed in the respective PEM-resistant cell lines. Quantitative RT-PCR analysis revealed that, besides an increased gene expression of thymidylate synthase in PEM-resistant PC-9 cells, the solute carrier family 19 member1 (SLC19A1) gene expression was markedly decreased in PEM-resistant A549 cells. The siRNA-mediated knockdown of SLC19A1 endowed the parental cell lines with PEM resistance. Conversely, PEM-resistant PC-9 cells carrying an epidermal growth factor receptor (EGFR) mutation acquired resistance to a tyrosine kinase inhibitor erlotinib. Although erlotinib can inhibit the phosphorylation of EGFR and Erk, it is unable to suppress the phosphorylation of Akt in PEM-resistant PC-9 cells. Additionally, PEM-resistant PC-9 cells were less sensitive to the PI3K inhibitor LY294002 than parental PC-9 cells. These results indicate that SLC19A1 negatively regulates PEM resistance in NSCLC, and that EGFR-tyrosine-kinase-inhibitor resistance was acquired with PEM resistance through Akt activation in NSCLC harboring EGFR mutations.

Contrasting effects of cyclophosphamide on anti-CTL-associated protein 4 blockade therapy in two mouse tumor models.

Immune checkpoint blockade is a promising anticancer therapy, but must be used in combination with other anticancer therapies to increase its therapeutic efficacy. Cyclophosphamide (CP) is a chemotherapeutic drug that shows immune-modulating effects. In this study, we examined the effect of CP on anti-CTL-associated protein 4 (CTLA-4) blockade therapy in two mouse tumor models. Drastic tumor regression was observed in the CT26 colon carcinoma model after i.p. injection of CP (100 mg/kg) followed by anti-CTLA-4 antibody. However, administration in the reverse order increased apoptosis in tumor-specific CD8+ T cells. In the RENCA renal carcinoma model, the antitumor effect of combination therapy was marginal and the tumor-bearing state reduced body weight with an increased serum level of interleukin-6. Interestingly, although CP monotherapy increased myeloid-derived suppressor cells (MDSCs) in the spleens of both models, subsequent anti-CTLA-4 therapy increased MDSCs only in RENCA-bearing mice. Additionally, the serum levels of chemokine ligand 2 and C-X-C motif chemokine 10 were increased by the combination therapy only in RENCA-bearing mice and in vivo depletion of Gr-1+ cells augmented the antitumor effect to some degree. These results reveal a contrasting effect of CP on anti-CTLA-4 therapy between the two mouse tumor models. Cyclophosphamide augments the antitumor effect of anti-CTLA-4 therapy in CT26-bearing hosts, whereas CP after anti-CTLA-4 therapy attenuates this effect through induction of apoptosis in tumor-reactive T cells. Alternatively, CP-induced MDSCs can be increased by anti-CTLA-4 therapy only in RENCA-bearing hosts with an elevated level of interleukin-6.

HIF-2α dictates the susceptibility of pancreatic cancer cells to TRAIL by regulating survivin expression.

Cancer cells develop resistance to therapy by adapting to hypoxic microenvironments, and hypoxia-inducible factors (HIFs) play crucial roles in this process. We investigated the roles of HIF-1α and HIF-2α in cancer cell death induced by tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) using human pancreatic cancer cell lines. siRNA-mediated knockdown of HIF-2α, but not HIF-1α, increased susceptibility of two pancreatic cancer cell lines, Panc-1 and AsPC-1, to TRAIL in vitro under normoxic and hypoxic conditions. The enhanced sensitivity to TRAIL was also observed in vivo. This in vitro increased TRAIL sensitivity was observed in other three pancreatic cancer cell lines. An array assay of apoptosis-related proteins showed that knockdown of HIF-2α decreased survivin expression. Additionally, survivin promoter activity was decreased in HIF-2α knockdown Panc-1 cells and HIF-2α bound to the hypoxia-responsive element in the survivin promoter region. Conversely, forced expression of the survivin gene in HIF-2α shRNA-expressing Panc-1 cells increased resistance to TRAIL. In a xenograft mouse model, the survivin suppressant YM155 sensitized Panc-1 cells to TRAIL. Collectively, our results indicate that HIF-2α dictates the susceptibility of human pancreatic cancer cell lines, Panc-1 and AsPC-1, to TRAIL by regulating survivin expression transcriptionally, and that survivin could be a promising target to augment the therapeutic efficacy of death receptor-targeting anti-cancer therapy.

Hypoxia-inducing factor (HIF)-1α-derived peptide capable of inducing cancer-reactive cytotoxic T lymphocytes from HLA-A24+ patients with renal cell carcinoma.

Hypoxic tumor microenvironment makes cancer cells to be therapy-resistant and hypoxia-inducing factors (HIFs) play a central role in hypoxic adaptation. Especially, renal cell carcinoma (RCC) is often associated with von Hippel-Lindau (VHL) gene mutations, leading to up-regulation of HIFs. However, from a different point of view, this suggests the possibility that HIFs could be promising targets in anti-cancer therapy. In this study, we searched for HIF-1α-derived peptides that are able to induce RCC-reactive cytotoxic T lymphocytes (CTLs) from HLA-A24+ RCC patients. Among five peptides derived from HIF-1α, which were prepared based on the binding motif to the HLA-A24 allele, a HIF-1α278-287 peptide induced peptide-specific CTLs from peripheral blood mononuclear cells of HLA-A24+ RCC patients most effectively. In immunoblot assays, the expression of HIF-1α was lowly detected in whole and nuclear lysates of RCC cell lines even under normoxia (20% O2), and their expression in whole lysates was increased under hypoxia (1% O2). Additionally, HIF-1α278-287 peptide-stimulated T cells showed a higher cytotoxicity against HLA-A24+ HIF-1α-expressing RCC cells than against HLA-A24- HIF-1α-expressing RCC cells. The cytotoxicity was inhibited by the addition of HIF-1α278-287 peptide-pulsed cold target cells. Altogether, these results indicate that the HIF-1α278-287 peptide could be a candidate for peptide-based anti-cancer vaccines for HLA-A24+ RCC patients.

Age-associated impairment of antitumor immunity in carcinoma-bearing mice and restoration by oral administration of Lentinula edodes mycelia extract.

Because cancer is associated with aging, immunological features in the aged should be considered in anticancer immunotherapy. In this study, we investigated antitumor immunity in aged mice using a CT26 colon carcinoma model. The tumor growth of CT26 was accelerated in aged mice compared with that in young mice, but this difference was not observed in nude mice. The serum levels of IL-6 and TNF-α were higher in aged mice than those in young mice, irrespective of the CT26-bearing state. The in vitro induction of CT26-specific CTLs from aged mice that were vaccinated with doxorubicin (DTX)-treated CT26 cells was impaired. In vivo neutralization of IL-6, but not TNF-α, showed a tendency to restore the in vitro induction of CT26-specific CTLs from vaccinated aged mice. Analyses on tumor-infiltrating immune cells as early as day 5 after CT26 inoculation revealed that monocytic and granulocytic MDSCs preferentially infiltrated into tumor sites in aged mice compared with young mice. Alternatively, oral administration of Lentinula edodes mycelia (L.E.M.) extract, which has the potential to suppress inflammation in tumor-bearing hosts, decreased the serum levels of IL-6 in aged mice. When administration of L.E.M. extract was started 1 week earlier, CT26 growth was retarded in aged mice and the in vivo priming of tumor-specific CTLs was improved in CT26-vaccinated aged mice. These results indicate early infiltration of MDSCs is related to impaired immunity of aged hosts and that oral administration of L.E.M. extract can mitigate the impairment.

Detection of an Immunogenic HERV-E Envelope with Selective Expression in Clear Cell Kidney Cancer.

VHL-deficient clear cell renal cell carcinomas (ccRCC), the most common form of kidney cancer, express transcripts derived from the novel human endogenous retrovirus HERV-E (named CT-RCC HERV-E). In this study, we define a transcript encoding the entire envelope gene of HERV-E as expressed selectively in ccRCC tumors, as distinct from normal kidney tissues or other tumor types. Sequence analysis of this envelope transcript revealed long open reading frames encoding putative surface and transmembrane envelope proteins. Retroviral envelopes are known to be capable of eliciting immunity in humans. Accordingly, we found that HLA-A*0201-restricted peptides predicted to be products of the CT-RCC HERV-E envelope transcript-stimulated CD8(+) T cells, which could recognize HLA-A*0201-positive HERV-E-expressing kidney tumor cells. Overall, our results offer evidence of unique HERV-E envelope peptides presented on the surface of ccRCC cells, offering potentially useful tumor-restricted targets for T-cell-based immunotherapy of kidney cancer. Cancer Res; 76(8); 2177-85. ©2016 AACR.

Bcl-xL inhibition by molecular-targeting drugs sensitizes human pancreatic cancer cells to TRAIL.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) induces apoptosis in various types of cancer cells without damaging normal cells. However, in terms of pancreatic cancer, not all cancer cells are sensitive to TRAIL. In this study, we examined a panel of human pancreatic cancer cell lines for TRAIL sensitivity and investigated the effects of Bcl-2 family inhibitors on their response to TRAIL. Both ABT-263 and ABT-737 inhibited the function of Bcl-2, Bcl-xL, and Bcl-w. Of the nine pancreatic cancer cell lines tested, six showed no or low sensitivity to TRAIL, which correlated with protein expression of Bcl-xL. ABT-263 significantly sensitized four cell lines (AsPC-1, Panc-1, CFPAC-1, and Panc10.05) to TRAIL, with reduced cell viability and increased apoptosis. Knockdown of Bcl-xL, but not Bcl-2, by siRNA transfection increased the sensitivity of AsPC-1 and Panc-1 cells to TRAIL. ABT-263 treatment had no effect on protein expression of Bcl-2, Bcl-xL, or c-FLIPs. In Panc-1 cells, ABT-263 increased the surface expression of death receptor (DR) 5; the NF-κB pathway, but not endoplasmic reticulum stress, participated in the increase. In xenograft mouse models, the combination of TRAIL and ATB-737 suppressed the in vivo tumor growth of AsPC-1 and Panc-1 cells. These results indicate that Bcl-xL is responsible for TRAIL resistance in human pancreatic cancer cells, and that Bcl-2 family inhibitors could represent promising reagents to sensitize human pancreatic cancers in DR-targeting therapy.

Identification of Programmed Death Ligand 1-derived Peptides Capable of Inducing Cancer-reactive Cytotoxic T Lymphocytes From HLA-A24+ Patients With Renal Cell Carcinoma.

Molecular therapy targeting tumor angiogenesis has been the standard treatment for metastatic renal cell carcinoma (mRCC). However, despite their significant antitumor effects, most of patients with mRCC have not been cured. Under such circumstances, anticancer immunotherapy has been considered a promising treatment modality for mRCC, and cancer-reactive cytotoxic T lymphocytes (CTLs) are the most powerful effectors among several immune cells. However, anticancer CTLs can be inhibited by several immune inhibitory mechanisms, including the interaction between programmed death 1 (PD-1) and its ligand PD-L1, on T cells and cancer cells, respectively. Alternatively, this also means that PD-L1 could be a promising target for anticancer immunotherapy. Therefore, we searched for PD-L1-derived peptides that are applicable for anticancer vaccine for HLA-A24(+) RCC patients. Among 5 peptides derived from PD-L1, which were prepared based on the binding motif to the HLA-A24(+) allele, both PD-L1(11-19) and PD-L1(41-50) peptides induced peptide-specific CTLs from peripheral blood mononuclear cells of HLA-A24(+) RCC patients. Such PD-L1 peptide-stimulated CD8 T cells showed cytotoxicity against HLA-A24(+) and PD-L1-expressing RCC cells. Although IFN-γ treatment increased PD-L1 expression on PD-L1(low) RCC cells, their sensitivity to cytotoxicity of PD-L1 peptide-stimulated CD8(+) T cells varied between patients. Altogether, these results indicate that both PD-L1(11-19) and PD-L1(41-50) peptides could be candidates for peptide-based anticancer vaccines for HLA-A24(+) mRCC patients.

The Roles of ROS and Caspases in TRAIL-Induced Apoptosis and Necroptosis in Human Pancreatic Cancer Cells.

Death signaling provided by tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) can induce death in cancer cells with little cytotoxicity to normal cells; this cell death has been thought to involve caspase-dependent apoptosis. Reactive oxygen species (ROS) are also mediators that induce cell death, but their roles in TRAIL-induced apoptosis have not been elucidated fully. In the current study, we investigated ROS and caspases in human pancreatic cancer cells undergoing two different types of TRAIL-induced cell death, apoptosis and necroptosis. TRAIL treatment increased ROS in two TRAIL-sensitive pancreatic cancer cell lines, MiaPaCa-2 and BxPC-3, but ROS were involved in TRAIL-induced apoptosis only in MiaPaCa-2 cells. Unexpectedly, inhibition of ROS by either N-acetyl-L-cysteine (NAC), a peroxide inhibitor, or Tempol, a superoxide inhibitor, increased the annexin V-/propidium iodide (PI)+ early necrotic population in TRAIL-treated cells. Additionally, both necrostatin-1, an inhibitor of receptor-interacting protein kinase 1 (RIP1), and siRNA-mediated knockdown of RIP3 decreased the annexin V-/PI+ early necrotic population after TRAIL treatment. Furthermore, an increase in early apoptosis was induced in TRAIL-treated cancer cells under inhibition of either caspase-2 or -9. Caspase-2 worked upstream of caspase-9, and no crosstalk was observed between ROS and caspase-2/-9 in TRAIL-treated cells. Together, these results indicate that ROS contribute to TRAIL-induced apoptosis in MiaPaCa-2 cells, and that ROS play an inhibitory role in TRAIL-induced necroptosis of MiaPaCa-2 and BxPC-3 cells, with caspase-2 and -9 playing regulatory roles in this process.

New polycomb group protein enhancer of zeste homolog (EZH) 2-derived peptide with the potential to induce cancer-reactive cytotoxic T lymphocytes in prostate cancer patients with HLA-A3 supertype alleles.

Analyses on reactivity of anti-cancer cytotoxic T lymphocytes (CTLs) and clinical application of peptide-based anti-cancer vaccine have been mainly focused on patients with HLA-A2 or -A24 alleles. In this study, we identified an enhancer of zeste homolog (EZH) 2-derived peptide applicable for anti-cancer vaccine for prostate cancer patients with HLA-A3 supertype alleles. Five EZH2-derived peptides that were prepared based on the binding motif to the HLA-A3 supertype alleles (HLA-A11, -A31, and -A33) were functionally screened for their potential to induce peptide-specific CTLs from peripheral blood mononuclear cells (PBMCs) of HLA-A3 supertype allele(+) prostate cancer patients. As a result, EZH2733-741 peptide was found to efficiently induce peptide-specific CTLs. The EZH2733-741 peptide-stimulated and purified CD8(+) T cells from PBMCs of HLA-A3 supertype allele(+) prostate cancer patients showed higher cytotoxicity against HLA-A3 supertype allele-expressing LNCaP prostate cancer cells than against parental LNCaP cells. This cytotoxicity against HLA-A3 supertype allele-expressing LNCaP cells was partially but significantly inhibited by the addition of EZH2733-741 peptide-pulsed competitive cells. These results indicate that the EZH2733-741 peptide could be a promising candidate for peptide-based immunotherapy for HLA-A3 supertype allele(+) prostate cancer patients.

Intermittent chemotherapy can retain the therapeutic potential of anti-CD137 antibody during the late tumor-bearing state.

Immunomodulating monoclonal antibodies (mAb) can evoke antitumor T-cell responses, which are attenuated by regulatory T cells (Treg) and myeloid-derived suppressor cells (MDSC). Treatment with cyclophosphamide (CP) and gemcitabine (GEM) can mitigate the immunosuppression by Treg and MDSC, respectively. In the current study, we examined the antitumor effects of a combination of local injection with anti-CD137 mAb and intermittent low-dose chemotherapy using CP and GEM in subcutaneously established CT26 colon carcinoma. Although a significant antitumor effect was observed when local anti-CD137 mAb therapy (5 µg) was started early in the tumor-bearing stage (day 10), no therapeutic efficacy was observed when the mAb therapy was started at a later tumor-bearing stage (day 17). Analyses of the tumor-infiltrating immune cells revealed that the number of Gr-1(high/low) CD11b(+) MDSC started to increase 13 days after tumor inoculation, whereas injection with low-dose (50 mg/kg) CP and GEM mitigated this increase. In addition, although intermittent injections with low-dose CP and GEM on days 10 and 18 suppressed tumor growth significantly, additional local injections of anti-CD137 mAb on days 19, 21, and 23 further augmented the therapeutic efficacy. Cytotoxic T lymphocytes reactive to CT26 and a tumor antigen peptide were induced successfully from the spleen cells of tumor-cured or tumor-stable mice. In a bilateral tumor inoculation model, this combination therapy achieved systemic therapeutic effects and suppressed the growth of mAb-untreated tumors. These results suggest that intermittent immunochemotherapy using CP and GEM could retain the therapeutic potential of anti-CD137 mAb that is normally impaired during the late tumor-bearing stage.

Bcl-2 family inhibition sensitizes human prostate cancer cells to docetaxel and promotes unexpected apoptosis under caspase-9 inhibition.

Docetaxel (DTX) is a useful chemotherapeutic drug for the treatment of hormone-refractory prostate cancer. However, emergence of DTX resistance has been a therapeutic hurdle. In this study, we investigated the effect of combining DTX with Bcl-2 family inhibitors using human prostate cancer cell lines (PC3, LNCaP, and DU145 cells). PC3 cells were less sensitive to DTX than were the other two cell lines. In contrast to ABT-199, which inhibits Bcl-2 and Bcl-w, both ABT-263 and ABT-737, which inhibit Bcl-2, Bcl-xL, and Bcl-w, significantly augmented the antitumor effect of DTX on PC3 cells. ABT-263 also enhanced the antitumor effect of DTX on a DTX-resistant PC3 variant cell line. The antitumor effect of ABT-263 was due mainly to its inhibitory effect on Bcl-xL. In a xenograft mouse model, DTX and ABT-737 combination therapy significantly inhibited PC3 tumor growth. Interestingly, although ABT-263 activated caspase-9 in PC3 cells, inhibition of caspase-9 unexpectedly promoted ABT-263-induced apoptosis in a caspase-8-dependent manner. This augmented apoptosis was also observed in LNCaP cells. These findings indicate that Bcl-xL inhibition can sensitize DTX-resistant prostate cancer cells to DTX, and they reveal a unique apoptotic pathway in which antagonism of Bcl-2 family members in caspase-9-inhibited prostate cancer cells triggers caspase-8-dependent apoptosis.

Transfection of poly(I:C) can induce reactive oxygen species-triggered apoptosis and interferon-ß-mediated growth arrest in human renal cell carcinoma cells via innate adjuvant receptors and the 2-5A system.

BACKGROUND: Synthetic double-stranded RNA poly(I:C) is a useful immune adjuvant and exhibits direct antitumor effects against several types of cancers. In this study, we elucidated the mechanisms underlying the effects induced in poly(I:C)-transfected human renal cell carcinoma (RCC) cells. RESULTS: In contrast to the lack of an effect of adding poly(I:C), poly(I:C) transfection drastically decreased RCC cell viability. Poly(I:C) transfection induced reactive oxygen species (ROS)-dependent apoptosis in RCC cells and decreased the mitochondrial membrane potential (ΔΨm). Treatment with N-acetyl-L-cysteine (NAC), a ROS scavenger, suppressed apoptosis and restored the ΔΨm. Although the levels of phosphorylated γH2A.X, an indicator of DNA damage, increased in poly(I:C)-transfected RCC cells, NAC treatment decreased their levels, suggesting ROS-mediated DNA damage. Furthermore, poly(I:C) transfection increased the levels of phosphorylated p53, NOXA, and tBid. Immunoblots and assays with a panel of caspase inhibitors revealed that poly(I:C) transfection-induced apoptosis was dependent on caspase-8 and -9, as well as caspase-2. Alternatively, poly(I:C) transfection increased mRNA expression of interferon (IFN)-ß, and treatment with IFN-ß suppressed growth of RCC cells without apoptosis. In addition, cyclinD1 and c-Myc expression decreased in poly(I:C)-transfected RCC cells. Moreover, RNA interference experiments revealed that poly(I:C) transfection exerted apoptotic effects on RCC cells through innate adjuvant receptors and the 2-5A system, the latter of which induces apoptosis in virus-infected cells. CONCLUSIONS: These results suggest that poly(I:C) transfection induced two types of effects against RCC cells such as apoptosis, as a result of ROS-mediated DNA damage, and IFN-ß-mediated growth arrest, both of which were exerted via innate adjuvant receptors and the 2-5A system.

Identification of erythropoietin receptor-derived peptides having the potential to induce cancer-reactive cytotoxic T lymphocytes from HLA-A24(+) patients with renal cell carcinoma.

Molecular targeting therapy with anti-angiogenic agents, including sunitinib and sorafenib, has been proven to be the first- and second-line standard treatments for metastatic renal cell carcinoma (mRCC) worldwide. Despite their significant antitumor effects, most of the patients with mRCC have not been cured. Under such circumstances, anti-cancer immunotherapy has been considered as a promising treatment modality for mRCC, and cytotoxic T lymphocytes (CTLs) are the most powerful effectors among several immune cells and molecules. Therefore, we previously conducted anti-cancer vaccine therapy with peptides derived from carbonic anhydrase-9 and vascular endothelial growth factor receptor-1 as phase-I/II trials for mRCC patients and reported their clinical benefits. Alternatively, up-regulated expression of erythropoietin (Epo) and its receptor (EpoR) in RCC has been reported, and their co-expression is involved in tumorigenesis. In order to increase options for peptide-based vaccination therapy, we searched for novel EpoR-peptides for HLA-A24(+) RCC patients. Among 5 peptides derived from EpoR, which were prepared based on the binding motif to the HLA-A24 allele, EpoR52-60 peptide had the potential to induce peptide-specific CTLs from peripheral blood mononuclear cells of HLA-A24(+) RCC patients. Cytotoxicity toward HLA-A24(+) and EpoR-expressing RCC cells was ascribed to peptide-specific CD8(+) T cells. These results indicate that the EpoR52-60 peptide could be a promising candidate for a peptide-based anti-cancer vaccine for HLA-A24(+) mRCC patients.

Pifithrin-µ, an inhibitor of heat-shock protein 70, can increase the antitumor effects of hyperthermia against human prostate cancer cells.

Hyperthermia (HT) improves the efficacy of anti-cancer radiotherapy and chemotherapy. However, HT also inevitably evokes stress responses and increases the expression of heat-shock proteins (HSPs) in cancer cells. Among the HSPs, HSP70 is known as a pro-survival protein. In this study, we investigated the sensitizing effect of pifithrin (PFT)-µ, a small molecule inhibitor of HSP70, when three human prostate cancer cell lines (LNCaP, PC-3, and DU-145) were treated with HT (43°C for 2 h). All cell lines constitutively expressed HSP70, and HT further increased its expression in LNCaP and DU-145. Knockdown of HSP70 with RNA interference decreased the viability and colony-forming ability of cancer cells. PFT-µ decreased the viabilities of all cell lines at one-tenth the dose of Quercetin, a well-known HSP inhibitor. The combination therapy with suboptimal doses of PFT-µ and HT decreased the viability of cancer cells most effectively when PFT-µ was added immediately before HT, and this combination effect was abolished by pre-knockdown of HSP70, suggesting that the effect was mediated via HSP70 inhibition. The combination therapy induced cell death, partially caspase-dependent, and decreased proliferating cancer cells, with decreased expression of c-Myc and cyclin D1 and increased expression of p21(WAF1/Cip), indicating arrest of cell growth. Additionally, the combination therapy significantly decreased the colony-forming ability of cancer cells compared to therapy with either alone. Furthermore, in a xenograft mouse model, the combination therapy significantly inhibited PC-3 tumor growth. These findings suggest that PFT-µ can effectively enhance HT-induced antitumor effects via HSP70 inhibition by inducing cell death and arrest of cell growth, and that PFT-µ is a promising agent for use in combination with HT to treat prostate cancer.

Butyric acid attenuates intestinal inflammation in murine DSS-induced colitis model via milk fat globule-EGF factor 8.

Butyric acid, a short-chain fatty acid and one of the main metabolites of intestinal microbial fermentation of dietary fiber, has been shown to have an important role in maintaining the integrity of the intestinal mucosa, while it also has been shown to exert potent anti-inflammatory effects both in vitro and in vivo. However, the precise mechanisms underlying those effects have not been fully identified. We exposed colonic epithelial cells to butyric acid, then extracted total RNA samples, and subsequently hybridized them to microarray chips. Among the upregulated genes, milk fat globule-epidermal growth factor 8 (MFG-E8) was elevated by approximately fivefold. We previously reported that the potential therapeutic benefits of MFG-E8 in intestinal tissue injury were dependent not only on enhanced clearance of apoptotic cells but also required diverse cellular events for maintaining epithelial integrity. The influence of butyric acid on cell function is often attributed to its inhibition of histone deacetylases (HDACs). We found that acetylation on histone 3 lysine 9 (acetyl-H3K9) around the MFG-E8 promoter was significantly increased with butyric acid exposure. Experimental colitis was induced by administration of dextran sodium sulfate (DSS) in C57BL/6N (MFG-E8+/+) and MFG-E8-/- mice. Although the colonic bacterial compositions in wild-type (WT) and MFG-E8-/- mice were not significantly different, intrarectal administration of butyric acid during an acute phase of colitis attenuated intestinal inflammatory parameters and inhibited body weight loss in the WT mice. Our novel findings suggest that butyric acid has significant anti-inflammatory effects partly via MFG-E8 on DSS-induced murine experimental colitis.

The HSP70 and autophagy inhibitor pifithrin-µ enhances the antitumor effects of TRAIL on human pancreatic cancer.

TRAIL and agonistic death receptor-specific antibodies can induce apoptosis in cancer cells with little cytotoxicity to normal cells. To improve TRAIL-induced antitumor effects, we tested its effectiveness in combination with pifithrin (PFT)-µ, which has the potential to inhibit HSP70 function and autophagy, both of which participate in TRAIL resistance in cancer cells. Among the four human pancreatic cancer cell lines tested, MiaPaca-2, Panc-1, and BxPC-3 cells showed varying sensitivities to TRAIL. In MiaPaca-2 and Panc-1 cells, knockdown of HSP70 or beclin-1, the latter an autophagy-related molecule, by RNA interference augmented TRAIL-induced antitumor effects, decreasing cell viability, and increasing apoptosis. On the basis of these findings, we next determined whether the TRAIL-induced antitumor effects could be augmented by its combination with PFT-µ. The combination of TRAIL plus PFT-µ significantly decreased the viability and colony-forming ability of MiaPaca-2 and Panc-1 cells compared with cells treated with either agent alone. When applied alone, PFT-µ increased Annexin V(+) cells in both caspase-dependent and -independent manners. It also promoted TRAIL-induced apoptosis and arrested cancer cell growth. Furthermore, PFT-µ antagonized TRAIL-associated NF-κB activation in cancer cells. In a xenograft mouse model, combination therapy significantly inhibited MiaPaca-2 tumor growth compared with treatment with either agent alone. The results of this study suggest protective roles for HSP70 and autophagy in TRAIL resistance in pancreatic cancer cells and suggest that PFT-µ is a promising agent for use in therapies intended to enhance the antitumor effects of TRAIL.

Metronomic chemotherapy with low-dose cyclophosphamide plus gemcitabine can induce anti-tumor T cell immunity in vivo.

Several chemotherapeutic drugs have immune-modulating effects. For example, cyclophosphamide (CP) and gemcitabine (GEM) diminish immunosuppression by regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs), respectively. Here, we show that intermittent (metronomic) chemotherapy with low-dose CP plus GEM can induce anti-tumor T cell immunity in CT26 colon carcinoma-bearing mice. Although no significant growth suppression was observed by injections of CP (100 mg/kg) at 8-day intervals or those of CP (50 mg/kg) at 4-day intervals, CP injection (100 mg/kg) increased the frequency of tumor peptide-specific T lymphocytes in draining lymph nodes, which was abolished by two injections of CP (50 mg/kg) at a 4-day interval. Alternatively, injection of GEM (50 mg/kg) was superior to that of GEM (100 mg/kg) in suppressing tumor growth in vivo, despite the smaller dose. When CT26-bearing mice were treated with low-dose (50 mg/kg) CP plus (50 mg/kg) GEM at 8-day intervals, tumor growth was suppressed without impairing T cell function; the effect was mainly T cell dependent. The metronomic combination chemotherapy cured one-third of CT26-bearing mice that acquired tumor-specific T cell immunity. The combination therapy decreased Foxp3 and arginase-1 mRNA levels but increased IFN-γ mRNA expression in tumor tissues. The percentages of tumor-infiltrating CD45(+) cells, especially Gr-1(high) CD11b(+) MDSCs, were decreased. These results indicate that metronomic chemotherapy with low-dose CP plus GEM is a promising protocol to mitigate totally Treg- and MDSC-mediated immunosuppression and elicit anti-tumor T cell immunity in vivo.

Combining a peptide vaccine with oral ingestion of Lentinula edodes mycelia extract enhances anti-tumor activity in B16 melanoma-bearing mice.

New anticancer vaccines must overcome regulatory T cell (Treg)-mediated immunosuppression. We previously reported that oral ingestion of Lentinula edodes mycelia (L.E.M.) extract restores melanoma-reactive T cells in melanoma-bearing mice via a mitigation of Treg-mediated immunosuppression. In this study, we investigated the effect of oral ingestion of the extract on peptide vaccine-induced anti-tumor activity. The day after subcutaneous inoculation in the footpad with B16 melanoma, mice were freely fed the extract and were vaccinated with a tyrosinase-related protein 2(180-188) peptide. The peptide vaccine was repeated thrice weekly. Melanoma growth was significantly suppressed in mice treated with both the peptide vaccine and L.E.M. extract compared with mice treated with vaccine or extract alone, and the effect was CD8(+) T cell-dependent. The combination therapy increased H-2K(b)-restricted and B16 melanoma-reactive T cells in the draining lymph nodes and spleen. Flow cytometric and immunohistological analyses revealed that the combination therapy significantly decreased the percentage of Tregs in the draining lymph nodes and spleen of melanoma-bearing mice compared to treatment with vaccine or extract alone. Kinetic analyses of peptide-specific T cells and Tregs revealed that induction of peptide-specific T cells by the peptide vaccine alone was transient, but when combined with L.E.M. extract, it efficiently prolonged the duration of peptide-specific T cell induction without increasing the percentage of Tregs. These results indicate that combination therapy enhances peptide vaccine-induced anti-tumor activity due to attenuation of the increase in the percentage of Tregs in tumor-bearing hosts.

Antitumor effects of cytoplasmic delivery of an innate adjuvant receptor ligand, poly(I:C), on human breast cancer.

Innate adjuvant receptors are expressed in immune cells and some types of cancers. If antitumor therapies targeting these receptors are established, it is likely that they will be therapeutically beneficial because antitumor effects and immune-cell activation can be induced simultaneously. In this study, we tested this possibility of using an innate adjuvant receptor ligand, polyinosinic-polycytidylic acid [poly(I:C)], to treat human breast cancer cell lines. Three breast cancer cell lines (MCF-7, MDA-MB-231, and BT-549) were used in this study. Poly(I:C) was transfected into these cancer cells to stimulate melanoma differentiation-associated gene (MDA) 5, which is a cytoplasmic adjuvant receptor. Poly(I:C) transfection significantly reduced the viability of all cell lines in a manner partially dependent on MDA5. Flow cytometeric analyses and immunoblot assays revealed that the antitumor effect depended on both caspase-dependent apoptosis and c-Myc- and cyclinD1-dependent growth arrest. Interestingly, poly(I:C) transfection was accompanied by autophagy, which is thought to protect cancer cells from apoptosis after poly(I:C) transfection. In a xenograft mouse model, local transfection of poly(I:C) significantly inhibited the growth of xenografted MDA-MB-231 cells. Our findings indicate that cytoplasmic delivery of poly(I:C) can induce apoptosis and growth arrest of human breast cancer cells, and that therapy-associated autophagy prevents apoptosis. The results of this study suggest that the innate adjuvant receptors are promising targets and that their ligands could serve as antitumor reagents, which have the potential to simultaneously induce antitumor effects and activate immune cells.