Hypoxia-related carbonic anhydrase 9 induces serpinB9 expression in cancer cells and apoptosis in T cells via acidosis.

Hypoxia is a common feature of solid tumors. However, the impact of hypoxia on immune cells within tumor environments remains underexplored. Carbonic anhydrase 9 (CA9) is a hypoxia-responsive tumor-associated enzyme. We previously noted that regardless of human CA9 (hCA9) expression, hCA9-expressing mouse renal cell carcinoma RENCA (RENCA/hCA9) presented as a "cold" tumor in syngeneic aged mice. This study delves into the mechanisms behind this observation. Gene microarray analyses showed that RENCA/hCA9 cells exhibited elevated mouse serpinB9, an inhibitor of granzyme B, relative to RENCA cells. Corroborating this, RENCA/hCA9 cells displayed heightened resistance to antigen-specific cytotoxic T cells compared with RENCA cells. Notably, siRNA-mediated serpinB9 knockdown reclaimed this sensitivity. In vivo tests showed that serpinB9 inhibitor administration slowed RENCA tumor growth, but this effect was reduced in RENCA/hCA9 tumors, even with adjunctive immune checkpoint blockade therapy. Further, inducing hypoxia or introducing the mouse CA9 gene upregulated serpinB9 expression, and siRNA-mediated knockdown of the mouse CA9 gene inhibited the hypoxia-induced induction of serpinB9 in the original RENCA cells. Supernatants from RENCA/hCA9 cultures had lower pH than those from RENCA, suggesting acidosis. This acidity enhanced serpinB9 expression and T cell apoptosis. Moreover, coculturing with RENCA/hCA9 cells more actively prompted T cell apoptosis than with RENCA cells. Collectively, these findings suggest hypoxia-associated CA9 not only boosts serpinB9 in cancer cells but also synergistically intensifies T cell apoptosis via acidosis, characterizing RENCA/hCA9 tumors as "cold."

T-cell responses and combined immunotherapy against human carbonic anhydrase 9-expressing mouse renal cell carcinoma.

Renal cell carcinoma (RCC) is known to respond to immune checkpoint blockade (ICB) therapy, whereas there has been limited analysis of T-cell responses to RCC. In this study, we utilized human carbonic anhydrase 9 (hCA9) as a model neoantigen of mouse RENCA RCC. hCA9-expressing RENCA RCC (RENCA/hCA9) cells were rejected in young mice but grew in aged mice. CD8+ T cells were the primary effector cells involved in rejection in young mice, whereas CD4+ T cells participated at the early stage. Screening of a panel of hCA9-derived peptides revealed that mouse CD8+ T cells responded to hCA9288-296 peptide. Mouse CD4+ T cells responded to lysates of RENCA/hCA9, but not RENCA cells, and showed reactivity to hCA9 276-290, which shares three amino acids with hCA9 288-296 peptide. Immunohistochemistry analysis revealed that few T cells infiltrated RENCA/hCA9 tissues in aged mice. ICB therapy of anti-PD-1/anti-CTLA-4 antibodies promoted T-cell infiltration into tumor tissues, whereas no definite antitumor effect was observed. However, additional combination with cyclophosphamide or axitinib, a vascular endothelial growth factor receptor inhibitor, induced complete regression in half of the RENCA/hCA9-bearing aged mice with increased expression of PD-L1 in tumor tissues. These results indicate that hCA9 can be a useful model neoantigen to investigate antitumor T-cell responses in mice with RCC, and that RENCA/hCA9 in aged mice can serve as a non-inflamed 'cold' tumor model facilitating the development of effective combined immunotherapies for RCC.

Immunogenic chemotherapy in two mouse colon cancer models.

Aside from the induction of cell death, some anticancer chemotherapeutic drugs can modulate antitumor immune responses. In this study, we examined the anticancer effects of 5-fluorouracil (5-FU) and oxaliplatin (L-OHP), which are standard chemotherapeutic drugs for colon cancer, combined with cyclophosphamide (CP) in two mouse colon cancer models (CT26 and MC38 colon adenocarcinoma models). In the CT26 model, two injections of 5-FU/L-OHP and CP significantly suppressed the growth of subcutaneously established CT26 tumors compared with either 5-FU/L-OHP or CP, without a significant loss of body weight. The anticancer effect was weakened in nude mice. Cured mice acquired protective immunity against CT26, and CT26-specific cytotoxic T cells (CTLs) were induced from their spleen cells. Analysis of tumor-infiltrating immune cells revealed that 5-FU/L-OHP treatment with or without CP increased the proportion of CD8+ T cells at tumor sites. The 5-FU/L-OHP treatment decreased the proportion of granulocytic myeloid-derived suppressor cells (MDSCs) and increased monocytic MDSCs in tumor sites, whereas the addition of CP treatment reversed these changes. In the MC38 model, although significant anticancer effects of the triple combination therapy were seen, additional treatment with anti-PD-1 antibody increased the number of cured mice. These mice exhibited protective immunity against MC38, and MC38-specific CTLs were generated from their spleen cells. Together, these results indicate that the antitumor effects of the combination of 5-FU/L-OHP and CP mainly depend on host T cells; moreover, the therapeutic efficacy can be effectively boosted by immune checkpoint blockade.

Supplementation of l-arginine boosts the therapeutic efficacy of anticancer chemoimmunotherapy.

Myeloid-derived suppressor cells (MDSCs) play a crucial role in immunosuppression in tumor-bearing hosts. MDSCs express arginase-I and indoleamine 2,3-dioxygenase; they suppress T-cell function by reducing the levels of l-arginine and l-tryptophan, respectively. We examined the anticancer effects of supplementation of these amino acids in CT26 colon carcinoma-bearing mice. Oral supplementation of l-arginine or l-tryptophan (30 mg/mouse) did not affect tumor growth, whereas oral supplementation of d-arginine was lethal. Supplementation of l-arginine showed a tendency to augment the efficacy of cyclophosphamide (CP). CP reduced the proportions of granulocytic MDSCs and increased the proportions of monocytic MDSCs in the spleen and tumor tissues of CT26-bearing mice. l-Arginine supplementation alone did not affect the MDSC subsets. CP treatment tended to reduce the plasma levels of l-arginine in CT26-bearing mice and significantly increased the number of tumor-infiltrating CD8+ T cells. In addition, l-arginine supplementation significantly increased the proportions of tumor peptide-specific CD8+ T cells in draining lymph nodes. Importantly, additional supplementation of l-arginine significantly increased the number of cured mice that were treated with CP and anti-PD-1 antibody. Totally, l-arginine supplementation shows promise for boosting the therapeutic efficacy of chemoimmunotherapy.

Pemetrexed sensitizes human lung cancer cells to cytotoxic immune cells.

Pemetrexed (PEM) is a useful drug that can be combined with immune checkpoint blockade therapy for treatment of patients with advanced non-small-cell lung cancer (NSCLC). However, its effects on anti-cancer immunity, especially the sensitivity of NSCLC cells to cytotoxic immune cells, have not been fully investigated. In this study, we examined the effects of PEM on the sensitivity of human NSCLC cells to two different types of cytotoxic immune cells. Pre-treatment with PEM increased the sensitivity of two NSCLC cell lines, PC9 and A549, to activated T cells and natural killer (NK) cells, and decreased the expression of anti-apoptotic proteins, including XIAP and Mcl-1. In addition, PEM treatment increased the cell surface expression of programmed death-ligand 1 (PD-L1) on PC9 cells. PEM-induced upregulation of PD-L1 on PC9 cells was at least partially ascribed to activation of ERK and the NFκB pathway. In contrast, PEM treatment increased the expression of UL16-binding proteins (ULBP), ligands for the NKG2D NK receptor, on PC9 and A549 cells, as well as the induction of senescence. Although the addition of anti-programmed cell death 1 antibody showed no effect on the sensitivity of PEM-treated PC9 and A549 cells to activated T cells, that of anti-NKG2D antibody decreased the enhanced sensitivity of PEM-treated A549 cells to NK cells. These results indicate that PEM can effectively sensitize human NSCLC cells to cytotoxic immune cells while modulating the expression of immune-regulatory molecules.

Different sensitivities of senescent breast cancer cells to immune cell-mediated cytotoxicity.

Senescence is a state of growth arrest induced not only in normal cells but also in cancer cells by aging or stress, which triggers DNA damage. Despite growth suppression, senescent cancer cells promote tumor formation and recurrence by producing cytokines and growth factors; this state is designated as the senescence-associated secretory phenotype. In this study, we examined the susceptibility of senescent human breast cancer cells to immune cell-mediated cytotoxicity. Doxorubicin (DXR) treatment induced senescence in 2 human breast cancer cell lines, MDA-MB-231 and BT-549, with the induction of γH2AX expression and increased expression of p21 or p16. Treatment with DXR also induced the expression of senescence-associated ß-galactosidase and promoted the production of pro-inflammatory cytokines. Importantly, DXR-treated senescent MDA-MB-231 cells showed increased sensitivity to 2 types of immune cell-mediated cytotoxicity: cytotoxicity of activated CD4+ T cells and Ab-dependent cellular cytotoxicity by natural killer cells. This increased sensitivity to cytotoxicity was partially dependent on tumor necrosis factor-related apoptosis-inducing ligand and perforin, respectively. This increased sensitivity was not observed following treatment with the senescence-inducing cyclin-dependent kinase-4/6 inhibitor, abemaciclib. In addition, treatment with DXR, but not abemaciclib, decreased the expression of antiapoptotic proteins in cancer cells. These results indicated that DXR and abemaciclib induced senescence in breast cancer cells, but that they differed in their sensitivity to immune cell-mediated cytotoxicity. These findings could provide an indication for combining anticancer immunotherapy with chemotherapeutic drugs or molecular targeting drugs.

Endothelial cellular senescence is inhibited by liver X receptor activation with an additional mechanism for its atheroprotection in diabetes.

Senescence of vascular endothelial cells leads to endothelial dysfunction and contributes to the progression of atherosclerosis. Liver X receptors (LXRs) are nuclear receptors whose activation protects against atherosclerosis by transcriptional regulation of genes important in promoting cholesterol efflux and inhibiting inflammation. Here we found that LXR activation with specific ligands reduced the increase in senescence-associated (SA) ß-gal activity, a senescence marker, and reversed the decrease in telomerase activity, a replicative senescence marker, in human endothelial cells under high glucose. This effect of LXR activation was associated with reduced reactive oxygen species and increased endothelial NO synthase activity. A series of experiments that used siRNAs indicated that LXRß mediates the prevention of endothelial cellular senescence, and that sterol regulatory element binding protein-1, which was up-regulated as a direct LXRß target gene, may act as a brake of endothelial cellular senescence. Although oral administration of the LXR ligand led to severe fatty liver in diabetic rats, concomitant therapy with metformin avoided the development of hepatic steatosis. However, the preventive effect of the LXR ligand on SA ß-gal-stained cells in diabetic aortic endothelium was preserved even if metformin was coadministered. Taken together, our studies demonstrate that an additional mechanism, such as the regulation of endothelial cellular senescence, is related to the antiatherogenic properties of LXRs, and concomitant treatment with metformin may provide a clinically useful therapeutic strategy to alleviate an LXR activation-mediated adverse effects on liver triglyceride metabolism.

A novel JAK-STAT inhibitor, 2-[(3-Carbamoyl-2-thienyl)amino]-2-oxoethyl(2,6-dichlorophenyl)acetate, suppresses helper T cell differentiation in vitro and collagen-induced arthritis in vivo.

Th17 cells, which have been implicated in autoimmune diseases including rheumatoid arthritis (RA), require the JAK-STAT3 pathway for their differentiation and functions. Recently, JAK inhibitors have been developed as a therapeutic drug for RA. However, the current JAK inhibitors are not optimized to STAT3 compared with other STATs. In this study, we found a new lead compound of a small molecule JAK-STAT inhibitor, 2-[(3-Carbamoyl-2-thienyl)amino]-2-oxoethyl (2,6-dichlorophenyl)acetate, which inhibits STAT3 as efficiently as other STATs. This compound, named JI069, was selected by STAT3 reporter assay in combination with an in silico docking model. JI069 inhibited gp130 signaling by inducing dissociation between gp130 and JAK1. In HEK293T cells and primary T cells, JI069 suppressed STAT3 activation as efficiently as other STATs, including STAT1, STAT5, and STAT6. JI069 effectively suppressed Th1, Th2, and Th17 differentiation while strongly promoted iTreg differentiation. JI069 suppressed symptoms of the collagen-induced arthritis (CIA) model in mice, and inhibited the cytokine production from T cells as well as the STAT3 phosphorylation of synovial cells. These data suggest that JI069 is a new type of JAK inhibitor which has potential for the treatment of immunological disorders.

Th17 cells carrying TCR recognizing epidermal autoantigen induce psoriasis-like skin inflammation.

Psoriasis is considered a Th17-type autoimmune skin inflammatory disease; however, involvement of an autoantigen-specific TCR has not been established. In this study, we show that psoriasis-like skin inflammation can be induced by autoreactive Th17 cells. We previously developed the desmoglein 3-specific TCR-transgenic (Dsg3H1) mouse, in which CD4⁺ T cells recognize physiological epidermal autoantigen. T cells from Dsg3H1 mice were polarized into Th17 cells in vitro and then adoptively transferred into Rag2⁻/⁻ mice. Dsg3H1-Th17 cells induced severe psoriasis-like skin inflammation within 2 wk after transfer in the tissues in which desmoglein 3 is expressed. Such pathology was not observed when wild-type Th17 cells or Th1-skewed Dsg3H1 T cells were transferred, and it was strongly suppressed by anti-IL-12/23 and anti-IL-17 Abs. Although IFN-γ⁺/IL-17⁺ T cells accumulated in the skin lesions of mice that received Dsg3H1-Th17 cells, IFN-γ-deficient Dsg3H1-Th17 cells were fully pathogenic. These results demonstrate that cutaneous psoriasis-like immunopathology can be developed by epidermis-specific recognition of Th17 cells, which is strictly dependent on IL-17 but not IFN-γ.

Smad2/3 and IRF4 play a cooperative role in IL-9-producing T cell induction.

IL-9 is a pleiotropic cytokine that can regulate autoimmune and allergic responses. Th9 cells can develop from naive T cells or Th2 cells through stimulation by TGF-ß in vitro. In this study, we demonstrated that Smad2 and Smad3 are necessary for IL-9 production from T cells in an OVA-induced asthma model using T cell-specific Smad2- and Smad3-deficient mice. Smad2 and Smad3 were also redundantly essential for TGF-ß signaling to induce histone modifications for Il9 transcription. Although Smad2/3 was recruited to the Il9 promoter by TGF-ß stimulation, they are not sufficient to activate the Il9 promoter. By the screening the transcription factors, we found that IFN regulatory factor 4 (IRF4) was essential for the Smad2/3-mediated Il9 promoter activation. In addition, Smad2/3 physically interacted with IRF4, and Smad2/3 did not bind to the Il9 promoter and could not induce Th9 in IRF4-deficient T cells. Similarly, IRF4 could not stimulate Il9 transcription in the absence of Smad2/3, and TGF-ß enhanced IRF4 recruitment to the Il9 promoter in a Smad2/3-dependent manner. We propose that Smad2/3 and IRF4 cooperatively transactivate the Il9 promoter and play an important role in regulating allergic immune responses by inducing Th9 cells.

Retinoic acid receptor agonist activity of naturally occurring diterpenes.

Recent accumulating evidence indicates that all-trans retinoic acid (ATRA) may be useful for preventing or treating inflammation, allergy, and autoimmune diseases, despite its severe side effects. In this study, screening of 99 crude drugs for retinoic acid receptor (RAR) ligands by luciferase reporter assay demonstrated that the methanol extract of Aralia cordata Rhizoma most effectively activates the transcriptional activity of RARα. Pimaradienoic acid (ent-pimara-8(14),15-dien-19-oic acid) was subsequently isolated as the constituent capable of activating RAR. Pimaric acid and abietic acid, which have similar structures to pimaradienoic acid, were also found to be novel RAR agonists, although abietic acid only slightly activated peroxisome proliferator-activated receptor gamma. These three natural RAR agonists with diterpene structures, while structurally different from ATRA, were able to increase the mRNA levels of the constitutive androstane receptor in HepG2 cells, induce F9 cell differentiation followed by Cyp26a1 mRNA expression, and differentiate HL-60 cells via RAR activation in a different manner from ATRA. These results demonstrate that some diterpenes exist as naturally occurring RAR agonists and that the differences in chemical structure between ATRA and these diterpenes may induce distinct gene activation and a specific cellular response.

Rexinoids isolated from Sophora tonkinensis with a gene expression profile distinct from the synthetic rexinoid bexarotene.

The retinoid X receptor (RXR) plays a critical role in transcriptional regulation via formation of an RXR homodimer or heterodimers with partner nuclear receptors. Despite the numerous beneficial effects, only a limited number of naturally occurring RXR agonists are known. In this report, two prenylated flavanones (1 and 2) isolated from Sophora tonkinensis were identified as new rexinoids that preferentially activated RXRs, relative to the retinoic acid receptor. The activities of 1 and 2 were the most potent among naturally occurring rexinoids, yet 2 orders of magnitude lower than the synthetic rexinoid bexarotene. Compounds 1 and 2 activated particular RXR heterodimers in a manner similar to bexarotene. A microarray assay followed by quantitative real-time polymerase chain reaction analyses on RNAs isolated from C2C12 myotubes treated with 1 or 2 demonstrated that they significantly increased mRNA levels of lipoprotein lipase, angiopoietin-like protein 4, and heme oxygenase-1. In contrast, bexarotene preferentially potentiated transcription of genes involved in lipogenesis and lipid metabolism such as sterol regulatory element-binding protein-1, fatty acid synthase, and apolipoprotein D by a liver X receptor agonist. In this study, we have demonstrated that two newly identified naturally occurring rexinoids, 1 and 2, possess properties different from bexarotene.

Senolysis of gemcitabine-induced senescent human pancreatic cancer cells.

INTRODUCTION: Gemcitabine (GEM) is often used to treat pancreatic cancer. Many anti-cancer drugs induce cancer cell death, but some cells survive after cell cycle arrest. Such a response to DNA damage is termed cellular senescence. Certain drugs, including the Bcl-2-family inhibitor ABT-263, kill senescent cells; this is termed senolysis. In this study, we examined the therapeutic benefits of ABT-263 in GEM-induced senescence of human pancreatic cancer cells. METHODS AND RESULTS: Of four pancreatic cancer cell lines (PANC-1, AsPC-1, CFPAC-1, and PANC10.05), GEM induced senescent features in PANC-1 and AsPC-1 cells, including increases in the cell sizes and expression levels of mRNAs encoding interleukin (IL)-6/IL-8 and induction of ß-galactosidase. Successive treatment with GEM and ABT-263 triggered apoptosis in PANC-1 and AsPC-1 cells and suppressed colony formation significantly. Senolysis of GEM-induced senescent pancreatic cancer cells by ABT-263 was triggered by a Bcl-xL inhibitor, but not by a Bcl-2 inhibitor, suggesting a central role for Bcl-xL in senolysis. In a xenograft mouse model, combined treatment with GEM and ABT-737 (an ABT-263 analog exhibiting the same specificity) suppressed in vivo growth of AsPC-1 significantly. CONCLUSION: Together, our results indicate that sequential treatment with GEM and senolytic drugs effectively kill human pancreatic cancer cells.

Therapeutic Senolysis of Axitinib-Induced Senescent Human Lung Cancer Cells.

BACKGROUND: Tyrosine kinase inhibitors (TKIs) inhibit receptor-mediated signals in cells. Axitinib is a TKI with high specificity for vascular endothelial growth factor receptors (VEGFRs). AIM: We determined whether axitinib could induce senescence in human cancer cells and be lysed by the senolytic drug ABT-263. METHODS: Human lung and breast adenocarcinoma cell lines were used. These cells were cultured with axitinib or a multi-target TKI lenvatinib. The expression of ß-galactosidase, VEGFRs, Ki-67, reactive oxygen species (ROS) of cancer cells, and their BrdU uptake were evaluated by flow cytometry. The mRNA expression of p21 and IL-8 was examined by quantitative PCR. The effects of TKIs on phosphorylation of Akt and Erk1/2, as downstream molecules of VEGFR signaling, were examined by immunoblot. The in vivo anti-cancer effect was examined using a xenograft mice model. RESULTS: Axitinib, but not lenvatinib, induced cellular senescence (increased cell size and enhanced expression of ß-galactosidase) in all adenocarcinoma cell lines. Axitinib-induced senescence was unrelated to the expression of VEGFRs on cancer cells. ROS were involved in axitinib-induced senescence. Axitinib-induced senescent lung adenocarcinoma A549 cells were drastically lysed by ABT-263. In A549-xenografted mice, combination therapy with axitinib and ABT-263 significantly suppressed tumor growth with the induction of apoptotic cancer cells.

CDK inhibitors suppress Th17 and promote iTreg differentiation, and ameliorate experimental autoimmune encephalomyelitis in mice.

Th17 cells, which have been implicated in autoimmune diseases, require IL-6 and TGF-ß for early differentiation. Several Smad-independent pathways including the JNK and the RhoA-ROCK pathways have been implicated in the induction of RORγt, the master regulator of Th17, however, molecular mechanisms underlying Smad-independent pathway remain largely unknown. To identify novel pathways involved in Th17 differentiation, we screened 285 chemical inhibitors for known signaling pathways. Among them, we found that Kenpaullone, a GSK3-ß and CDK inhibitor, efficiently suppressed TGF-ß-mediated RORγt induction and enhanced Foxp3 induction in primary T cells. Another CDK inhibitor, Roscovitine, but not other GSK3-ß inhibitors, suppressed Th17 differentiation and enhanced iTreg development. Kenpaullone and Roscovitine suppressed experimental autoimmune encephalomyelitis (EAE), a typical Th17-mediated autoimmune disease model. These two compounds enhanced STAT5 phosphorylation and restored IL-2 production in the presence of TGF-ß. These data suggest that CDK inhibitors modulate TGF-ß-signaling pathways, which restore TGF-ß-mediated suppression of IL-2 production, thereby modifying the Th17/iTreg balance.

Methoxyflavones isolated from the whole plant of Scutellaria rubropunctata Hayata var. rubropunctata promote osteoblast differentiation in MC3T3-E1 cells.

In this study, we isolated two new methoxyflavones (1 and 2) and eight known methoxyflavones (3-10) from the whole plant of Scutellaria rubropunctata Hayata var. rubropunctata (SR). Based on spectroscopic analyses, the methoxyflavones were identified as 5,8,2',6'-tetramethoxy-6,7-methylenedioxyflavone (1) and 5,2',6'-trimethoxy-6,7-methylenedioxyflavone (2). We reported SR might have effects on promoting osteoblast differentiation and stimulating estrogen receptor (ER) in the previous study. Then, the effects of 1-10 on pre-osteoblast MC3T3-E1 cells were investigated, and 1, 2, and 9 were observed to promote alkaline phosphatase activity. To evaluate their effect on osteogenesis-related genes, we performed gene expression analysis using quantitative real-time PCR after treatment of MC3T3-E1 cells with these compounds. Although 2 was only effective at lower concentrations, 1 and 9 upregulated the mRNA levels of Runx2, Osterix, Osteopontin, Osteocalcin, Smad1, and Smad4. These results indicate that 1 and 9 may induce osteoblast differentiation by activating Runx2 via the BMP/Smad pathway and may play a central role in the promotion of osteoblast differentiation by SR. The ER agonist activity of 1-10 were tested using a luciferase reporter assay in HEK293 cells. However, none of the compounds exhibited remarkable activity. Thus, SR may contain other compounds that contribute to its ER agonist activity.

Preferential induction of Th17 cells in vitro and in vivo by Fucogalactan from Ganoderma lucidum (Reishi).

The mushroom known as Reishi (Ganoderma lucidum) has been used as an herbal medicine for tumor treatment and immune system activation. Because its effects on the differentiation of effector T helper cells have not yet been fully understood, we investigated the effects of Reishi and those of its principal ingredient, ß-glucan, on the activation of dendritic cells and the differentiation of Th17 cells. Reishi extracts as well as purified ß-glucan (Curdran) activated DCs and caused them to produce large amounts of IL-23. ß-glucan also enhanced and sustained the transcription of IL-23p19. The MEK-ERK signaling pathway positively regulates IL-23p19 transcription in ß-glucan-stimulated DCs. In a mixed leukocyte reaction, Reishi-stimulated DCs preferentially induced Th17 cells. Furthermore, orally-administrated Reishi increased the percentages of Th17 cells and the transcription levels of antimicrobial peptides. Our results show that Reishi and ß-glucan activate DCs to produce large amounts of IL-23, which induces Th17 differentiation both in vitro and in vivo.

A naturally occurring rexinoid, honokiol, can serve as a regulator of various retinoid x receptor heterodimers.

We investigated the properties of honokiol, a natural rexinoid, as a regulator of retinoid X receptor (RXR) heterodimers with various partner nuclear receptors (NRs), in comparison with those of the synthetic rexinoid bexarotene. Honokiol alone was hardly capable of activating peroxisome proliferator-activated receptor (PPAR) γ/RXR, RXR/liver X receptor (LXR), and RXR/vitamin D receptor (VDR) heterodimers, whereas it effectively potentiated their activation by agonists for the partner NRs of the RXR heterodimers. These findings were further supported by increased mRNA and protein levels for the respective NR target genes. Bexarotene alone activated PPARγ/RXR and RXR/LXR heterodimers, but not RXR/VDR heterodimers, and facilitated the activation of all three RXR heterodimers by the respective PPARγ, LXR, and VDR agonists. When the potencies of honokiol and bexarotene were compared, honokiol was able to serve as a subsidiary agonist in the activation of RXR heterodimers in a similar manner to bexarotene. However, it seemed to potentiate the activation of PPARγ/RXR heterodimers by the PPARγ agonist rosiglitazone more efficiently than bexarotene, and was a less potent RXR agonist than bexarotene. In conclusion, we have demonstrated that honokiol is a rexinoid that possesses distinct properties from bexarotene, and mainly has subsidiary roles in the activation of RXR heterodimers by potentiating the activation of RXR heterodimers by agonists for the partner NRs.

Hydroxychloroquine Promotes Bcl-xL Inhibition-induced Apoptosis in BxPC-3 Human Pancreatic Cancer Cells.

BACKGROUND/AIM: Anti-apoptotic proteins, including Bcl-2 and Bcl-xL, hinder cancer treatment, and several drugs targeting these molecules have been developed. One is ABT-263 (navitoclax), which targets Bcl-2, Bcl-xL, and Bcl-w. On the other hand, hydroxychloroquine (HCQ) has been used as a drug for malaria infection and autoimmune disease. HCQ can exert a similar effect as chloroquine with fewer adverse events. In addition, HCQ exerts antitumor activity. In the present study, the effects of HCQ on ABT-263-induced antitumor activities were examined using three human pancreatic cancer cell lines (PANC-1, MiaPaCa-2, and BxPC-3). MATERIALS AND METHODS: In vitro effects of HCQ and ABT-263 were examined by cell viability, colony-forming assays, and flow cytometry. Protein expression was determined by immunoblotting. In vivo effects of HCQ and ABT-263 were examined by a xenograft mice model. RESULTS: Combined treatment with HCQ and ABT-263 synergistically decreased the viability of only BxPC-3 cells. This synergistic effect was not observed when HCQ was combined with ABT-199, an inhibitor specific to Bcl-2. The combination of HCQ and ABT-263 induced caspase-dependent apoptosis. Protein expression of Bcl-xL was more highly expressed in BxPC-3 cells than in the other two cell lines, and the combination of HCQ with a Bcl-xL inhibitor or siRNA-mediated knockdown of Bcl-xL induced apoptosis in BxPC-3 cells. Combination therapy with HCQ and ABT 737, an ABT-263 analogue, suppressed the in vivo growth of BxPC-3 with transient body-weight loss. CONCLUSION: HCQ effectively promotes Bcl-xL inhibition-induced apoptosis in BxPC-3 human pancreatic cancer cells.

Effects of a Whole Plant Extract of Scutellaria rubropunctata var. rubropunctata on Bone Metabolism with Estrogen Receptor Activation.

We screened natural resources for estrogen receptor (ER)-activating and bone metabolism-promoting activities with the aim of finding potential treatments for osteoporosis. A screen of 1531 extracts from Ryukyu Arc plants using a luciferase reporter assay identified an 80% MeOH extract of Scutellaria rubropunctata var. rubropunctata (SRE) with dose-dependent ER transcription-promoting activity. Importantly, SRE had no proliferative effect on human breast cancer cells. SRE enhanced the ALP activity of pre-osteoblast MC3T3-E1 cells after 72 h in culture and slightly enhanced mineralization at 14 and 21 d. SRE did not significantly affect the TRAP activity of RAW264.7 cells. Gene expression analysis in MC3T3-E1 cells by quantitative real-time PCR revealed that SRE upregulated the mRNA levels of Runx2, Osterix (Osx), Osteopontin (Opn), Osteocalcin (Ocn), Smad1, Smad4, and Smad5 at 72 h, and those of Runx2, Osx, Smad1, Smad4, and Smad5 at 21 d of osteogenic induction. Analysis of the expression levels of osteogenic markers suggested that SRE may promote osteogenic differentiation by acting at the early stage of differentiation rather than at the late stage of differentiation. These results indicate that SRE activates ER and induces osteoblast differentiation by activating Runx2 and Osx through the BMP/Smad pathway, suggesting that SRE may be useful for the prevention and treatment of postmenopausal osteoporosis.