Alternative splicing of Apoptosis Stimulating Protein of TP53-2 (ASPP2) results in an oncogenic isoform promoting migration and therapy resistance in soft tissue sarcoma (STS).

BACKGROUND: Metastatic soft tissue sarcoma (STS) are a heterogeneous group of malignancies which are not curable with chemotherapy alone. Therefore, understanding the molecular mechanisms of sarcomagenesis and therapy resistance remains a critical clinical need. ASPP2 is a tumor suppressor, that functions through both p53-dependent and p53-independent mechanisms. We recently described a dominant-negative ASPP2 isoform (ASPP2κ), that is overexpressed in human leukemias to promote therapy resistance. However, ASPP2κ  has never been studied in STS.  MATERIALS AND METHODS: Expression of ASPP2κ was quantified in human rhabdomyosarcoma tumors using immunohistochemistry and qRT-PCR from formalin-fixed paraffin-embedded (FFPE) and snap-frozen tissue. To study the functional role of ASPP2κ in rhabdomyosarcoma, isogenic cell lines were generated by lentiviral transduction with short RNA hairpins to silence ASPP2κ expression. These engineered cell lines were used to assess the consequences of ASPP2κ silencing on cellular proliferation, migration and sensitivity to damage-induced apoptosis. Statistical analyses were performed using Student's t-test and 2-way ANOVA. RESULTS: We found elevated ASPP2κ mRNA in different soft tissue sarcoma cell lines, representing five different sarcoma sub-entities. We found that ASSP2κ mRNA expression levels were induced in these cell lines by cell-stress. Importantly, we found that the median ASPP2κ expression level was higher in human rhabdomyosarcoma in comparison to a pool of tumor-free tissue. Moreover, ASPP2κ levels were elevated in patient tumor samples versus adjacent tumor-free tissue within individual patients. Using isogenic cell line models with silenced ASPP2κ expression, we found that suppression of ASPP2κ enhanced chemotherapy-induced apoptosis and attenuated cellular proliferation. CONCLUSION: Detection of oncogenic ASPP2κ in human sarcoma provides new insights into sarcoma tumor biology. Our data supports the notion that ASPP2κ promotes sarcomagenesis and resistance to therapy. These observations provide the rationale for further evaluation of ASPP2κ as an oncogenic driver as well as a prognostic tool and potential therapeutic target in STS.

Unraveling the IGF System Interactome in Sarcomas Exploits Novel Therapeutic Options.

Aberrant bioactivity of the insulin-like growth factor (IGF) system results in the development and progression of several pathologic conditions including cancer. Preclinical studies have shown promising anti-cancer therapeutic potentials for anti-IGF targeted therapies. However, a clear but limited clinical benefit was observed only in a minority of patients with sarcomas. The molecular complexity of the IGF system, which comprises multiple regulators and interactions with other cancer-related pathways, poses a major limitation in the use of anti-IGF agents and supports the need of combinatorial therapeutic strategies to better tackle this axis. In this review, we will initially highlight multiple mechanisms underlying IGF dysregulation in cancer and then focus on the impact of the IGF system and its complexity in sarcoma development and progression as well as response to anti-IGF therapies. We will also discuss the role of Ephrin receptors, Hippo pathway, BET proteins and CXCR4 signaling, as mediators of sarcoma malignancy and relevant interactors with the IGF system in tumor cells. A deeper understanding of these molecular interactions might provide the rationale for novel and more effective therapeutic combinations to treat sarcomas.

Aberrant expression of N-glycolyl GM3 ganglioside is associated with the aggressive biological behavior of human sarcomas.

BACKGROUND: The aberrant expression of N-glycolyl GM3 ganglioside (NeuGcGM3) in patients with sarcomas was reevaluated by assessing the relation of this molecule with some clinicopathological features and overall survival (OS) of patients. METHODS: Fifty formalin-fixed and paraffin-embedded specimens from patients diagnosed with sarcomas were included. For the evaluation of NeuGcGM3, the 14F7 monoclonal antibody followed by a peroxidase avidin-biotin system was used. Clinicopathological features were obtained from patient records. Survival rates were estimated by the Kaplan-Meier method and compared with the log-rank test. For multivariate analyses, the Cox regression model was used to identify independent prognostic factors for OS. RESULTS: The majority of samples had high levels of NeuGcGM3 expression (66.0%) that showed statistical correlation with age (p = 0.014), TNM stage (p = 0.022), histological grade (p = 0.013) and proliferation rates (p = 0.012). In addition, a tendency for association with tumor depth (p = 0.070) was evidenced. In univariate survival analysis, TNM stage (p = 0.000), occurrence of metastasis (p = 0.000) and expression of NeuGcGM3 (p = 0.034) were significant prognostic factors for OS, while a tendency for association was evidenced for histological grade (p = 0.091). Among these variables, only the presence of metastasis (p = 0.001) was an independent prognostic factor on multivariate analysis. CONCLUSIONS: The present research suggests the evaluation of NeuGcGM3 expression as a complementary prognostic factor in sarcoma, although our results need to be validated in a larger series and prospective studies. Moreover, our results could support the use of this molecule as a target for immunotherapy.

Nilotinib as Coadjuvant Treatment with Doxorubicin in Patients with Sarcomas: A Phase I Trial of the Spanish Group for Research on Sarcoma.

Purpose: Nilotinib plus doxorubicin showed to be synergistic regarding apoptosis in several sarcoma cell lines. A phase I/II trial was thus designed to explore the feasibility of nilotinib as coadjuvant of doxorubicin by inhibiting MRP-1/P-gp efflux activity. The phase I part of the study is presented here.Patients and Methods: Nilotinib 400 mg/12 hours was administered in fixed dose from day 1 to 6, and doxorubicin on day 5 of each cycle. Three dose escalation levels for doxorubicin at 60, 65, and 75 mg/m2 were planned. Cycles were repeated every 3 weeks for a total of 4 cycles. Eligible subtypes were retroperitoneal liposarcoma, leiomyosarcoma, and unresectable/metastatic high-grade chondrosarcoma.Results: Thirteen patients were enrolled: 7 chondrosarcoma, 4 liposarcoma, and 2 leiomyosarcoma. In 46 cycles administered, the most relevant grade 3/4 adverse effects per patient were neutropenia 54%, febrile neutropenia 15%, and asthenia 8%. No cardiac toxicity was observed. Only one dose-limiting toxicity (febrile neutropenia) was reported in the third dose level. With regard to efficacy, 1 partial response (1 liposarcoma), 9 stable diseases (5 chondrosarcoma, 2 liposarcoma, 1 leiomyosarcoma), and 3 progressive diseases (2 chondrosarcoma and 1 leiomyosarcoma) were present. ABCB1 and ABCC1 RNA expression levels decreased by 58.47-fold and 1.47-fold, respectively, on day 5 of the cycle.Conclusions: Combination of MRP-1/P-gp inhibitor, nilotinib, as coadjuvant with doxorubicin is feasible; it appears not to add substantial toxicity compared with doxorubicin alone. Pharmacodynamic study supports this concept. The recommended dose for the phase II part for doxorubicin was 75 mg/m2 Clin Cancer Res; 24(21); 5239-49. ©2018 AACR.

Trabectedin and Eribulin: Where Do They Fit in the Management of Soft Tissue Sarcoma?

OPINION STATEMENT: Trabectedin and eribulin are two agents that have been recently approved for the treatment of specific soft tissue sarcoma subtypes. They have proved to be a much-needed line of additional treatment for patients with these rare tumors, but their activity remains admittedly modest in most cases. Further exploitation of these novel agents is likely to require a more granular understanding of the salient mechanisms of action. For example, if as some studies suggest, eribulin derives its benefit from restructuring of tumor vasculature to improve efficacy of subsequent lines of therapy, then patients may benefit from its use earlier in the treatment pathway. The sequencing of trabectedin with other agents is also worth examining. In a disease like myxoid liposarcoma, consideration should be given to using trabectedin before other salvage regimens like gemcitabine and docetaxel, given its tolerability and excellent efficacy against this sarcoma subtype. Also, to be further investigated is the use of trabectedin in sarcoma subtypes which were excluded from the phase III study, but in which activity has been documented in earlier trials and subsequent reports. Combinations of trabectedin with other agents, particularly doxorubicin, have been explored, but the data to date do not support the routine use of these regimens.

Hyperthermia adds to trabectedin effectiveness and thermal enhancement is associated with BRCA2 degradation and impairment of DNA homologous recombination repair.

The tetrahydroisoquinoline trabectedin is a marine compound with approved activity against human soft-tissue sarcoma. It exerts antiproliferative activity mainly by specific binding to the DNA and inducing DNA double-strand breaks (DSB). As homologous recombination repair (HRR)-deficient tumors are more susceptible to trabectedin, hyperthermia-mediated on-demand induction of HRR deficiency represents a novel and promising strategy to boost trabectedin treatment. For the first time, we demonstrate enhancement of trabectedin effectiveness in human sarcoma cell lines by heat and characterize cellular events and molecular mechanisms related to heat-induced effects. Hyperthermic temperatures (41.8 or 43°C) enhanced significantly trabectedin-related clonogenic cell death and G2/M cell cycle arrest followed by cell type-dependent induction of apoptosis or senescence. Heat combination increased accumulation of γH2AX foci as key marker of DSBs. Expression of BRCA2 protein, an integral protein of the HRR machinery, was significantly decreased by heat. Consequently, recruitment of downstream RAD51 to γH2AX-positive repair foci was almost abolished indicating relevant impairment of HRR by heat. Accordingly, enhancement of trabectedin effectiveness was significantly augmented in BRCA2-proficient cells by hyperthermia and alleviated in BRCA2 knockout or siRNA-transfected BRCA2 knockdown cells. In peripheral blood mononuclear cells isolated from sarcoma patients, increased numbers of nuclear γH2AX foci were detected after systemic treatment with trabectedin and hyperthermia of the tumor region. The findings establish BRCA2 degradation by heat as a key factor for a novel treatment strategy that allows targeted chemosensitization to trabectedin and other DNA damaging antitumor drugs by on-demand induction of HRR deficiency.

Molecular inhibition of prostaglandin E2 with GW627368X: Therapeutic potential and preclinical safety assessment in mouse sarcoma model.

Prostaglandin E2, the major COX-2 product, acts via 4 functionally distinct prostanoid receptors, EP(1-4). PGE-2, through its receptors, feeds back to positively increase COX-2 expression augmenting its own synthesis thereby driving angiogenesis, while suppressing apoptosis and innate immunity. In addition to the well characterized PGE2/EP4/cAMP/PKA/CREB, EP4 activation increases GSK3 phosphorylation via PI3K and Akt consequently reducing ß-catenin phosphorylation. EP4 induces angiogenesis by enhancing VEGF production via ERK activation. These effects of EP4 are asserted either directly or via EGFR transactivation depending on the type of cancer. In view of the safety concerns regarding long term use of COX-2 inhibitors and to find more effective alternatives, we evaluated the potential of EP4 prostanoid receptor as a target for treating cancer progression using a highly selective EP4 antagonist, 4-(4,9-diethoxy-1,3-dihydro-1-oxo-2H-benz[f]isoindol-2-yl)-N-(phenylsulfonyl)-benzeneacetamide. Oral administration of GW627368X showed significant tumor regression characterized by tumor reduction and induction of apoptosis. Reduction in prostaglandin E2 synthesis also led to reduced level of VEGF in plasma. Regulation of multiple pathways downstream of EP4 was evident by down regulation of COX-2, p-Akt, p-MAPK and p-EGFR. Considering wide distribution of the EP4 prostanoid receptor in major organs and the array of physiological processes it contributes to, the safety profile of the drug was analyzed. No major organ toxicity, immunosupression, behavioral change or change in blood parameters attributable to the drug was observed. The results assert the significance of EP4 prostanoid receptor as a therapeutic target as well as the safety of EP4 blockade by GW627368X.

Compositions and anti-tumor activity of Pyropolyporus fomentarius petroleum ether fraction in vitro and in vivo.

The chemical compositions and anti-tumor activities of the petroleum ether fraction (PE), from mushroom Pyropolyporus fomentarius, were studied. Upon gas chromatography-mass spectrometry (GC-MS) analysis, nine major constituents were identified in the fraction. In vitro, the PE showed cytotoxic activity against murine sarcoma S180 (S180) cells in a dose- and time-dependent manner, and the cytotoxic effects were associated with apoptosis. The mitochondrial membrane potential loss and the intracellular ROS generation were greatly increased in the Pyropolyporus fomentarius PE treated group, suggesting cell apoptosis, induced by the PE in S180 cells, might be mitochondria dependent and ROS mediated. Consistent with in vitro findings, the in vivo study showed that the Pyropolyporus fomentarius PE was also effective in inhibiting the tumor growth induced by S180 cells and had lower immune organ toxicity. We found that the Pyropolyporus fomentarius PE has significant anti-tumor activity and great potential in screening anti-tumor drugs.

Fufang Kushen injection inhibits sarcoma growth and tumor-induced hyperalgesia via TRPV1 signaling pathways.

Cancer pain is a deleterious consequence of tumor growth and related inflammation. Opioids and anti-inflammatory drugs provide first line treatment for cancer pain, but both are limited by side effects. Fufang Kushen injection (FKI) is GMP produced, traditional Chinese medicine used alone or with chemotherapy to reduce cancer-associated pain. FKI limited mouse sarcoma growth both in vivo and in vitro, in part, by reducing the phosphorylation of ERK and AKT kinases and BAD. FKI inhibited TRPV1 mediated capsaicin-induced ERK phosphorylation and reduced tumor-induced proinflammatory cytokine production. Thus, FKI limited cancer pain both directly by blocking TRPV1 signaling and indirectly by reducing tumor growth.

Inhibitory activities of Lignum Sappan extractives on growth and growth-related signaling of tumor cells.

AIM: To investigate the active constituents of Lignum Sappan (Caesalpinia sappan L.) on growth-related signaling and cell mitosis. METHOD: The influence of the ethyl acetate (EtOAc) extract of Lignum Sappan and its constituents on growth-related signaling were evaluated by a luciferase assay in cells stably-transfected with NF-κB, STAT1, or STAT3 responsive luciferase reporter plasmid. The inhibitory effect on the cell cycle was determined by flow cytometric analysis. The anti-tumor activities were assessed in vitro and in vivo. RESULTS: The EtOAc extract of Lignum Sappan had inhibitory activities on growth-related signaling and cell mitosis. Three major active compounds were sappanchalcone, brazilin, and butein. Sappanchalcone blocked cell cycle progression in the G2/M phase, brazilin inhibited TNFα/NF-κB signaling, while butein inhibited IL-6/STAT3 signaling, as well as TNFα/NF-κB signaling. The three compounds all demonstrated cytotoxic activities against human tumor cells in vitro. In a S180 tumor cell-bearing mice model, the anti-tumor efficacy of the EtOAc extract was better than the individual compounds acting alone. CONCLUSION: These results indicate that Lignum Sappan contains multiple active compounds with different antitumor activities, which act synergistically to enhance their anti-tumor effects. The EtOAc extract of Lignum Sappan may be better than individual active constituent as a novel medicine for the treatment of cancer.

Anti-angiogenic therapy, a new player in the field of sarcoma treatment.

Sarcomas encompass a heterogeneous family of mesenchymal malignancies. In metastatic disease improvement in outcome has been limited and there is a clear need for the development of new therapies. One potential target is angiogenesis, already an accepted target for treatment of more prevalent cancers. Multiple (pre)clinical studies focused on the role of angiogenesis and anti-angiogenic treatment in sarcomas. However, getting significant results is complicated due to the relatively small number of patients and the broad range of sarcoma subtypes. Recently, pazopanib has been approved for the treatment of advanced soft tissue sarcoma patients, which is an important step forward and paves the way for the introduction of anti-angiogenic treatment in sarcomas. However, more studies are needed to understand the biological mechanisms by which patients respond to angiogenic inhibitors and to detect markers of response. This review covers the knowledge that has been gained on the role of angiogenesis and anti-angiogenic therapy in sarcomas.

Targeted therapy by combined inhibition of the RAF and mTOR kinases in malignant spindle cell neoplasm harboring the KIAA1549-BRAF fusion protein.

BACKGROUND: Oncologic patients who are extreme responders to molecularly targeted therapy provide an important opportunity to better understand the biologic basis of response and, in turn, inform clinical decision making. Malignant neoplasms with an uncertain histologic and immunohistochemical characterization present challenges both on initial diagnostic workups and then later in management, as current treatment algorithms are based on a morphologic diagnosis. Herein, we report a case of a difficult to characterize sarcoma-like lesion for which genomic profiling with clinical next generation sequencing (NGS) identified the molecular underpinnings of arrested progression(stable disease) under combination targeted therapy within a phase I clinical trial. METHODS: Genomic profiling with clinical next generation sequencing was performed on the FoundationOne™ platform (Foundation Medicine, Cambridge MA). Histopathology and immunohistochemical studies were performed in the Department of Pathology, MD Anderson Cancer Center (Houston, TX). Treatment was administered in the context of a phase I clinical trial ClinicalTrials.gov Identifier: (NCT01187199). RESULTS: The histology of the tumor was that of a spindle cell neoplasm, grade 2 by FNCLCC standards. Immunohistochemical staining was positive for S100 and CD34. Genomic profiling identified the following alterations: a KIAA1549-BRAF gene fusion resulting from a tandem duplication event, a homozygous deletion of PTEN, and frameshift insertion/deletions in CDKN2A A68fs*51, SUFU E283fs*3, and MAP3K1 N325fs*3. The patient had a 25% reduction in tumor (RECIST v1.1) following combination therapy consisting of sorafenib, temsirolimus, and bevazicumab within a phase I clinical trial. CONCLUSIONS: The patient responded to combination targeted therapy that fortuitously targeted KIAA1549-BRAF and PTEN loss within a spindle cell neoplasm, as revealed by genomic profiling based on NGS. This is the first report of a tumor driven by a KIAA1549-BRAF fusion responding to sorafenib-based combination therapy.

Polysaccharide from Ganoderma atrum evokes antitumor activity via Toll-like receptor 4-mediated NF-κB and mitogen-activated protein kinase signaling pathways.

Ganoderma atrum has been used as a traditional Chinese medicine for centuries. In this study, the antitumor activity of a novel G. atrum polysaccharide (PSG-1) was investigated in vitro and in vivo using S180 tumor-bearing mice. The results showed that PSG-1 significantly inhibited the proliferation of S180 via the activation of macrophages in a dose-dependent manner. PSG-1-primed macrophages exhibited a higher tumoricidal activity than untreated macrophages. Administration of PSG-1 significantly inhibited the growth of transplantable sarcoma S180-bearing mice and increased macrophage phagocytosis and the levels of cytokines and nitride oxide. Expression of Toll-like receptor (TLR) 4 in the membrane was markedly increased in PSG-1-treated groups, suggesting that it may be a possible receptor for PSG-1. PSG-1 also promoted the translocation of the p65 subunit of NF-κB from cytosol to nucleus and the degradation of IκBα. Moreover, the phosphorylation of p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinases 1/2, and c-Jun N-terminal kinase in macrophages was improved by PSG-1 in a dose-dependent manner. Therefore, it is suggested that PSG-1 may elicit its antitumor effect by improving immune system functions through TLR4-mediated NF-κB and MAPK signaling pathways.

Inhibition of P-glycoprotein-mediated transport by S-adenosylmethionine and cynarin in multidrug-resistant human uterine sarcoma MES-SA/Dx5 cells.

Multidrug resistance (MDR) to anticancer chemotherapy is often mediated by the overexpression of the plasma membrane drug transporter P-glycoprotein (Pgp) encoded by multidrug resistance gene (MDR1). Various chemosensitizing agents are able to inhibit Pgp activity but their clinical application is limited by their toxicity. Furthermore, hepatotoxicity related to chemotherapy causes delays of treatment in cancer patients and often requires supplementation of anti-tumour therapy with hepatoprotective agents. In this in vitro study, we investigated the effectiveness of an endogenous hepatoprotective agent, S-adenosylmethionine (SAMe), and a natural hepatoprotective compound, Cynarin (Cyn), to inhibit Pgp activity in order to evaluate their potential use as chemosensitizing agents. Human doxorubicin (doxo) resistant uterine sarcoma cells (MES-SA/Dx5) expressing high levels of Pgp were treated with two hepatoprotectors at various concentrations (1, 5 and 10 microM) that are clinically achievable, in the presence or absence of three different concentrations of doxo (2, 4 and 8 microM). In order to evaluate the effects of both hepatoprotectors, we measured the intracellular accumulation and cytotoxicity of doxo, the cellular GSH level, ROS production and catalase (CAT) activity. We found that treatment with 2, 4 and 8 microM doxo in the presence of SAMe or Cyn significantly increased the doxo accumulation and cytotoxicity on MES-SA/Dx5 cells, when compared to control cells receiving doxo alone. Moreover, treatment with SAMe or Cyn significantly increased GSH content, greater than 80 percent and 60 percent, respectively) and CAT activity greater than 60 and 150 percent, respectively) in resistant cancer cells, while ROS production was below the values of corresponding untreated control cells. Our in vitro findings provide a rationale for the potential clinical use of these hepatoprotectors both as chemosensitizing agents, to reverse Pgp-mediated MDR, and as antioxidants to protect normal cells from chemotherapy-induced cytotoxixity.

Impact of hyperthermic isolated limb perfusion on tumour oxygenation in soft tissue sarcoma.

PURPOSE: Hyperthermic isolated limb perfusion (ILP) with recombinant tumour necrosis factor alpha (TNF) and melphalan contributes to limb-saving treatment in patients with locally advanced extremity soft tissue sarcoma (STS). This study was conducted to evaluate the dynamic changes of tumour oxygenation and temperature during ILP and their effects on treatment response. PATIENTS AND METHODS: Tumour oxygenation (pO(2)) and tumour temperature were measured by intratumourally placed O(2)-sensitive catheter electrodes in 34 patients who underwent ILP for locally advanced or recurrent STS. Tumour response to ILP was assessed by the fraction of tumour necrosis in the resection specimen. RESULTS: Mean tumour pO(2) prior to application of TNF and melphalan was 36 mm Hg (range: 2-116 mm Hg) and dropped significantly to 13 mm Hg (range: 0-67 mm Hg, p = 0.03) during ILP. Mean tumour tissue temperature increased from 34.4°C (range 32.4-36.4) to 38.5°C (range 34.1-40.4, p = 0.0001). The mean fraction of necrosis in the resection specimen was 65% (range 5-99). Only the tumour tissue temperature at the onset of ILP correlated with the extent of tumour necrosis (p = 0.01). CONCLUSION: ILP with TNF and melphalan induces severe oxygen deprivation in soft tissue sarcoma. However, changes in tumour oxygenation did not correlate with treatment response.

Thermal dose fractionation affects tumour physiological response.

PURPOSE: It is unknown whether a thermal dose should be administered using a few large fractions with higher temperatures or a larger number of fractions with lower temperatures. To evaluate this we assessed the effect of administering the same total thermal dose, approximately 30 CEM43T(90), in one versus three to four fractions per week, over 5 weeks. MATERIALS AND METHODS: Canine sarcomas were randomised to receive one of the hyperthermia fractionation schemes along with fractionated radiotherapy. Tumour response was based on changes in tumour volume, oxygenation, water diffusion quantified using MRI, and a panel of histological and immunohistochemical end points. RESULTS: There was a greater reduction in tumour volume and water diffusion at the end of therapy in tumours receiving one hyperthermia fraction per week. There was a weak but significant association between improved tumour oxygenation 24 h after the first hyperthermia treatment and extent of volume reduction at the end of therapy. Finally, the direction of change of HIF-1α and CA-IX immunoreactivity after the first hyperthermia fraction was similar and there was an inverse relationship between temperature and the direction of change of CA-IX. There were no significant changes in interstitial fluid pressure, VEGF, vWF, apoptosis or necrosis as a function of treatment group or temperature. CONCLUSIONS: We did not identify an advantage to a three to four per week hyperthermia prescription, and response data pointed to a one per week prescription being superior.

Immunomodulatory effect of polysaccharides from submerged cultured Cordyceps gunnii.

CONTEXT: The genus Cordyceps (Clavicipitaceae) is a group of entomopathogenic fungi that is widely used as tonic food or invigorant with broad-spectrum medicinal properties in China. Cordyceps gunnii (Berk.)Berk (C. gunnii), is also well known as the Chinese rare caterpillar fungus and has similar pharmacological activities with Cordyceps sinensis (C. sinensis). Polysaccharides (PS) from various Cordyceps species have demonstrated many interesting biological activities, including antitumor, immunopotentiation, hypoglycemic, and hypocholesterolemic activities. OBJECTIVE: To investigate the effect of C. gunnii PS on the immunostimulatory antitumor function and expression of immune related cytokines in normal, immuno-suppressive, and H22-bearing mice, respectively. METHODS: C. gunnii PS were extracted with hot water at 80°C for 2 h. Normal, immuno-suppressive, and H22-bearing mice were treated with PS respectively. By detecting the value of macrophage phagocytic index, proliferation of lymphocytes, natural killer (NK) cell activity and expression of related cytokines, interleukin (IL-4), tumor necrosis factor-α (TNF-a) and interferon-γ (IFN-γ), and tumor inhibition index in H22-bearing mice additionally, the effect of PS on immunostimulatory antitumor function and its mechanism were studied. RESULTS: The total sugar content of the PS was determined to be 95% after purification. PS markedly increased the thymus and spleen indexes, the macrophage phagocytosis, the proliferation of splenic cells, and the level of IFN-γ and TNF-α. In tumor growth inhibition test, PS showed remarkable inhibition effects. CONCLUSION: PS from the C. gunnii could enhance nonspecific immunological function, humoral immunity, cellular immunity in mice, and inhibit tumor growth.

Antitumor activity and macrophage nitric oxide producing action of medicinal herb, Crassocephalum crepidioides.

BACKGROUND: Crassocephalum crepidioides, a plant distributed in Okinawa Islands, is known in folk medicine; however, its anticancer activity has not been investigated. The aim of this study was to determine the in vitro and in vivo antitumor activities of C. crepidioides on murine Sarcoma 180 (S-180) and related molecular mechanisms. METHODS: The antitumor effect of C. crepidioides was evaluated in S-180-cell-bearing mice. Cell growth was assessed using a colorimetric assay. Nitrite and nitrate levels were measured by colorimetry. The expression levels of inducible NO synthase (iNOS) in murine RAW264.7 macrophages was assessed by reverse transcriptase-polymerase chain reaction. Activation of iNOS promoter was detected by reporter gene. Activation of nuclear factor-κB (NF-κB) was evaluated by electrophoretic mobility shift assay. The role of NF-κB signaling was analyzed using inhibitors of NF-κB and dominant-negative mutants, and Western blot analysis. RESULTS: C. crepidioides extract delayed tumor growth in S-180-bearing mice. However, it did not inhibit S-180 cell growth in vitro. Supernatant of cultured C. crepidioides-stimulated RAW264.7 macrophages was cytotoxic to S-180 cells. This cytotoxicity was associated with nitric oxide (NO) production. NF-κB signaling pathway was crucial for the transcriptional activation of iNOS gene. Isochlorogenic acid, a component of C. crepidioides, induced NF-κB activation and iNOS expression. CONCLUSIONS: The results highlight the oncolytic and immunopotentiation properties of C. crepidioides mediated through NF-κB-induced release of NO from macrophages.

Beneficial effects of curcumin on antitumor activity and adverse reactions of doxorubicin.

Many medicines used in cancer chemotherapy decrease the quality of life (QOL). It is believed that an increase in food intake during cancer chemotherapy may produce an improvement in QOL. Curcumin is widely used as a coloring and flavoring agent in food. The effects of curcumin in relation to the chemotherapeutic drug doxorubicin (DOX) were examined. While DOX alone did not decrease tumor weight, the combination of DOX and curcumin significantly reduced tumor weight to 56.5% (p<0.05) of that of the control group. The combined curcumin enhanced apoptosis by DOX and decreased cell viability. The curcumin-DOX combination also suppressed activation of caspase-3, -8, and -9 compared to DOX alone. It is presumed that combining curcumin increased DOX-induced antitumor activity by suppressing the main caspase pathway and activating the main caspase independent pathway. The combination of curcumin and DOX suppressed the reduction of glutathione peroxidase activity and increased lipid peroxide levels in the heart. Therefore, it is expected that curcumin may reduce the adverse reactions associated with DOX. Our results suggest that curcumin can be used as a modulator to enhance the therapeutic index of cancer patients and improve their QOL.

Geranyl derivative of phloroacetophenone induces cancer cell-specific apoptosis through Bax-mediated mitochondrial pathway in MCF-7 human breast cancer cells.

Plant-derived polyhenols inhibit cancer cell proliferation and induce apoptosis. Recently, prenylflavonoids and alkyl-phloroacetophenones have been reported for their in vitro antitumor activity. In the present study, we examined the cytotoxic activity of prenyl (3-PAP) and geranyl (3-GAP) derivatives of phloroacetophenone, and xanthohumol (XN), a prenyl-chalcone, in human breast cancer (MCF-7) and human sarcoma (HT1080) cell lines in vitro. 3-GAP showed the strongest cytotoxicity in these cell lines with IC(50) values of less than 10 µM. In addition, we report that 3-GAP is a more potent anti-cancer agent for breast cancer than XN which is a well-known anticancer flavonoid. Moreover, 3-GAP did not induce cytotoxicity in the normal cell line, TCMK-1, whereas 3-PAP and XN significantly reduced TCMK-1 cell viability. In 3-GAP-treated MCF-7 cells, nuclear accumulation and transcriptional activity of p53 were increased. In addition, pro-apoptotic Bax but not B-cell lymphoma 2 (Bcl-2) expression was increased by 3-GAP. In accordance with the Bax increase, 3-GAP induced mitochondrial cytochrome c release and activated caspase-9, an initiator of the caspase cascade. In the MCF-7 cell line which does not express caspase-3, activation of caspase-7, a member of the caspase-3 subfamily, was increased by 3-GAP. The present results indicate that synthetic 3-GAP is a safe and effective anti-cancer agent, and the Bax-mediated mitochondrial pathway is the main apoptosis signaling pathway of 3-GAP in MCF-7 cells.