Repurposing nitric oxide donating drugs in cancer therapy through immune modulation.

BACKGROUND: Nitric oxide-releasing drugs are used for cardiovascular diseases; however, their effects on the tumor immune microenvironment are less clear. Therefore, this study explored the impact of nitric oxide donors on tumor progression in immune-competent mice. METHODS: The effects of three different nitric oxide-releasing compounds (SNAP, SNP, and ISMN) on tumor growth were studied in tumor-bearing mouse models. Three mouse tumor models were used: B16F1 melanoma and LL2 lung carcinoma in C57BL/6 mice, CT26 colon cancer in BALB/c mice, and LL2 lung carcinoma in NOD/SCID mice. After nitric oxide treatment, splenic cytokines and lymphocytes were analyzed by cytokine array and flow cytometry, and tumor-infiltrating lymphocytes in the TME were analyzed using flow cytometry and single-cell RNA sequencing. RESULTS: Low doses of three exogenous nitric oxide donors inhibited tumor growth in two immunocompetent mouse models but not in NOD/SCID immunodeficient mice. Low-dose nitric oxide donors increase the levels of splenic cytokines IFN-γ and TNF-α but decrease the levels of cytokines IL-6 and IL-10, suggesting an alteration in Th2 cells. Nitric oxide donors increased the number of CD8+ T cells with activation gene signatures, as indicated by single-cell RNA sequencing. Flow cytometry analysis confirmed an increase in infiltrating CD8+ T cells and dendritic cells. The antitumor effect of nitric oxide donors was abolished by depletion of CD8+ T cells, indicating the requirement for CD8+ T cells. Tumor inhibition correlated with a decrease in a subtype of protumor macrophages and an increase in a subset of Arg1-positive macrophages expressing antitumor gene signatures. The increase in this subset of macrophages was confirmed by flow cytometry analysis. Finally, the combination of low-dose nitric oxide donor and cisplatin induced an additive cancer therapeutic effect in two immunocompetent animal models. The enhanced therapeutic effect was accompanied by an increase in the cells expressing the gene signature of NK cell. CONCLUSIONS: Low concentrations of exogenous nitric oxide donors inhibit tumor growth in vivo by regulating T cells and macrophages. CD8+ T cells are essential for antitumor effects. In addition, low-dose nitric oxide donors may be combined with chemotherapeutic drugs in cancer therapy in the future.

Measurement of the refractive index of lenses from the maximum phase difference of the total internal reflection with a polarization camera.

The proposed total internal reflection (TIR)-based technique can be used for measuring the refractive index of lenses. Distribution of the phase difference between the s- and p-polarization states of the reflected light induced by TIR can be obtained by a polarization camera. The refractive index of the lens can be determined from the detected maximum phase difference, with the specific measurement equation. Only the maximum phase difference needs to be measured. Information about the incident angle, thickness of the lens, and the matching liquid is not needed. The experimental results demonstrate that the resolution of the system can reach 4.8×10-4RIU.

PODXL2 maintains cellular stemness and promotes breast cancer development through the Rac1/Akt pathway.

The cluster of differentiation 34 (CD34) family, which includes CD34, podocalyxin-like protein 1 (PODXL), and PODXL2, are type-I transmembrane sialomucins and markers of hematopoietic stem cells (HSCs) and vascular-associated tissues. CD34 family proteins are expressed by endothelial cells and hematopoietic precursors. PODXL is well known to be associated with invadopodia formation and to promote the epithelial-mesenchymal transition, tumor migration and invasion. PODXL expression was correlated with poor survival of cancer patients. However, the role of PODXL2 in cancer has been less fully explored. To reveal the novel role of PODXL2 in breast cancer, the present study evaluated PODXL2 levels in relation to clinical outcomes of cancer patients by performing a bioinformatics analysis using the Oncomine database, Kaplan-Meier plots, and the CCLE database. Empirical validation of bioinformatics predictions was conducted utilizing the short hairpin (sh)-RNA silencing method for PODXL2 in the BT474 invasive ductal breast carcinoma cell line. The bioinformatics analysis revealed that PODXL2 overexpression was correlated with poor survival of breast cancer patients, suggesting an oncogenic role of PODXL2 in breast carcinoma. In a validation experiment, knockdown of PODXL2 in BT474 cells slightly influenced cell proliferation, suppressed migration, and inhibited expressions of downstream molecules, including Ras-related C3 botulinum toxin substrate 1 (Rac1), phosphorylated (p)-Akt (S473), and p-paxillin (Y31) proteins. In addition, knockdown of PODXL2 reduced expression levels of cancer stem cell (CSC) markers, including Oct-4 and Nanog, and the breast CSC marker aldehyde dehydrogenase 1 (ALDH1). Collectively, our present study demonstrated that PODXL2 plays a crucial role in cancer development and could serve as a potential prognostic biomarker in breast cancer patients.

Gene signatures and potential therapeutic targets of amino acid metabolism in estrogen receptor-positive breast cancer.

Increased activity of amino acid transporters has been observed in a wide variety of cancers. However, whether amino acid metabolism is related to estrogen receptor-positive (ER+) breast cancer has been less well studied. We identified the rate-limiting enzyme involved in amino acid metabolism associated with ER+ breast cancer by integrating numerous bioinformatics tools and laboratory studies. The bioinformatics analysis revealed that highly expressed genes in ER+ breast cancer patients were correlated with breast cancer-related pathways, including ESR1 and PI3K signaling. The metabolic signaling and the amino acid metabolism were significantly regulated in breast neoplasms. We used the ER+ breast cancer cell line MCF-7 and breast cancer tissue from National Cheng Kung University Hospital to validate our findings in bioinformatics. In estradiol-treated MCF-7 cells, genes associated with anabolic metabolism of serine and methionine and genes associated with catabolic metabolism of tyrosine, phenylalanine and arginine were upregulated. Furthermore, the expression levels of ARG2, PSAT1, PSPH, TH, PAH, and MAT1A mRNA were increased in breast cancer patients relative to controls. The aforementioned genes were also found to be highly correlated with distant metastasis-free survival in breast cancer patients. High expression levels of ARG2, CBS, PHGDH, AHCY, HAL, TDO2, SHMT2, MAT1A, MAT2A, GLDC, GLS2, BCAT2, GLUD1, PAH and MTR contributed to poor prognoses, whereas high mRNA expression levels of HECA, CTH, PRODH, TAT, and MAT2B were correlated with good prognoses. FDA-approved drugs, including piperlongumine, ellipticine, etidronic acid, harmine, and meclozine, may have novel therapeutic effects in ER+ patients based on connectivity map (CMap) analyses. Collectively, our present study demonstrated that amino acid metabolism genes play crucial roles in tumor development and may serve as prospective drug targets or biomarkers for ER+ breast cancer.

Homoharringtonine induced immune alteration for an Efficient Anti-tumor Response in Mouse Models of Non-small Cell Lung Adenocarcinoma Expressing Kras Mutation.

Homoharringtonine (HHT), an inhibitor of protein synthesis, has been used to treat leukemia. Its therapeutic effects on non-small cell lung adenocarcinoma carrying KRAS mutation and their immune system are less understood. The present study examined the therapeutic efficacy and the immune effects of HHT in two murine lung tumor models, xenograft and transgenic, carrying the Kras mutation G12D and G12C respectively. HHT exhibited efficient anticancer activity, significantly suppressing lung tumor growth in vitro and in vivo. The levels of 22 cytokines and chemokines in splenocytes of tumor-bearing mice were examined. Interleukin-12 expression was lower in splenocytes of HHT-treated mice when compared to the controls as demonstrated by a cytokine array and an enzyme-linked immunosorbent assay. The expression levels of CD80, CD86, and CD69 in B220+ B cells from splenocytes of HHT-treated mice were higher than that of control mice in two mouse tumor models. Furthermore, antitumor effect of HHT was attenuated with depletion of B cells. Increased numbers of CD80+ and CD86+ B cells were observed in the mice treated with narciclasine, another translation inhibitor. In conclusion, HHT changed the features of immune cells, and exhibited efficient anti-tumor activity against lung tumor carrying mutant Kras expression.

Skin Delivery of Clec4a Small Hairpin RNA Elicited an Effective Antitumor Response by Enhancing CD8+ Immunity In Vivo.

Clec4a has been reported to be an immune suppressor of dendritic cells (DCs), but its potential role in cancer therapy remains to be elucidated. The present study investigated whether downregulating the expression of Clec4a via skin delivery of small hairpin RNA (shRNA) using a gene gun produced stronger host immunity and inhibited tumor progression in animal models. Administration of Clec4a2 shRNA delayed tumor growth in both mouse bladder and lung tumor-bearing mouse models. The result was further confirmed with a compensation experiment showing that the antitumor effects induced by Clec4a2 shRNA were restored by co-injection of a plasmid expressing exogenous Clec4a2. Increased numbers of infiltrating CD4+ and CD8+ T cells at tumor sites were observed in mice treated with Clec4a2 shRNA. Splenocytes from mice with Clec4a2 shRNA administration exhibited stronger cytotoxic activity compared with splenocytes from control mice. CD8-deletion in vivo abrogated the antitumor effects elicited by Clec4a2 shRNA. Additionally, shClec4a enhanced the antitumor effects of the Neu DNA vaccine in the MBT-2 tumor model. In summary, the findings provide evidence that silencing of Clec4a2 expression via skin delivery of shRNA produces an effective antitumor response and that Clec4a2 shRNA may have therapeutic potential as an adjuvant for cancer immunotherapy.

PSMB5 plays a dual role in cancer development and immunosuppression.

Tumor progression and metastasis are dependent on the intrinsic properties of tumor cells and the influence of microenvironment including the immune system. It would be important to identify target drug that can inhibit cancer cell and activate immune cells. Proteasome ß subunits (PSMB) family, one component of the ubiquitin-proteasome system, has been demonstrated to play an important role in tumor cells and immune cells. Therefore, we used a bioinformatics approach to examine the potential role of PSMB family. Analysis of breast TCGA and METABRIC database revealed that high expression of PSMB5 was observed in breast cancer tissue and that high expression of PSMB5 predicted worse survival. In addition, high expression of PSMB5 was observed in M2 macrophages. Based on our bioinformatics analysis, we hypothesized that PSMB5 contained immunosuppressive and oncogenic characteristics. To study the effects of PSMB5 on the cancer cell and macrophage in vitro, we silenced PSMB5 expression with shRNA in THP-1 monocytes and MDA-MB-231 cells respectively. Knockdown of PSMB5 promoted human THP-1 monocyte differentiation into M1 macrophage. On the other hand, knockdown PSMB5 gene expression inhibited MDA-MB-231 cell growth and migration by colony formation assay and boyden chamber. Collectively, our data demonstrated that delivery of PSMB5 shRNA suppressed cell growth and activated defensive M1 macrophages in vitro. Furthermore, lentiviral delivery of PSMB5 shRNA significantly decreased tumor growth in a subcutaneous mouse model. In conclusion, our bioinformatics study and functional experiments revealed that PSMB5 served as novel cancer therapeutic targets. These results also demonstrated a novel translational approach to improve cancer immunotherapy.

Mucin 2 silencing promotes colon cancer metastasis through interleukin-6 signaling.

Downregulation of Mucin 2 (MUC2) expression is associated with early carcinogenesis events in colon cancer. MUC2 plays a role in the progression of colon cancer, and reduced MUC2 protein expression correlates with increased interleukin-6 (IL-6) expression. However, the interaction between MUC2 and IL-6 in colorectal cancer metastasis remains unclear. We systematically analyzed MUC2 and IL-6 expression and determined the survival of cancer patients with high or low MUC2 and IL-6 expression using the Oncomine and PrognoScan databases, respectively. This analysis identified downregulation of MUC2 and overexpression of IL-6 in colon cancer but not in normal colon tissue, and this expression pattern was correlated with poor survival of colon cancer patients. We examined the effects of MUC2 on colon cancer metastasis and used vector-mediated application of short hairpin RNA (shRNA) to suppress MUC2 expression. MUC2 suppressed the migration of colon cancer cells in vitro and dramatically diminished liver metastases in vivo. Treatment with IL-6 increased signal transducer and activator of transcription 3 (STAT3) phosphorylation, promoted checkpoint kinase 2 (Chk2) activation, attenuated cAMP response element-binding protein (CREB) phosphorylation, and suppressed E-cadherin protein expression in MUC2-silenced HT-29 cancer cells. Most importantly, MUC2 is a potential prognostic indicator for colon cancer.

Argininosuccinate lyase interacts with cyclin A2 in cytoplasm and modulates growth of liver tumor cells.

Arginine is a critical amino acid in specific cancer types including hepatocellular carcinoma (HCC) and melanoma. Novel molecular mechanisms and therapeutic targets in arginine metabolism-mediated cancer formation await further identification. Our laboratory has previously demonstrated that arginine metabolic enzyme argininosuccinate lyase (ASL) promoted HCC formation in part via maintenance of cyclin A2 protein expression and arginine production for channeling to nitric oxide synthase. In this study, we investigated the mechanism by which ASL regulates cyclin A2 expression. We found that ASL interacted with cyclin A2 in HCC cells and the localization of their interaction was in the cytoplasm. Mutation of essential residues for enzymatic activity of ASL did not affect the binding of ASL to cyclin A2. Moreover, the mutant ASL retained the ability to restore the decreased tumorigenicity caused by ASL shRNA. Furthermore, overexpression of ASL conferred resistance to arginine deprivation therapy. Finally, the important pathways and potential therapeutic targets in ASL-regulated HCC were identified by bioinformatics analyses with Metacore database and Connectivity Map database. Our analyses suggested that bisoprolol, celecoxib, and ipratropium bromide, are potential therapeutics for ASL-regulated HCC formation. Thus, ASL interacts with cyclin A2 in cytoplasm, and may promote HCC formation through this non-enzymatic function. Overexpression of ASL may be a contributing factor in drug resistance for arginine deprivation therapy.

Cancer stem cell marker CD90 inhibits ovarian cancer formation via ß3 integrin.

Cancer stem cell (CSC) markers have been identified for CSC isolation and proposed as therapeutic targets in various types of cancers. CD90, one of the characterized markers in liver and gastric cancer, is shown to promote cancer formation. However, the underexpression level of CD90 in ovarian cancer cells and the evidence supporting the cellular mechanism have not been investigated. In the present study, we found that the DNA copy number of CD90 is correlated with mRNA expression in ovarian cancer tissue and the ovarian cancer patients with higher CD90 have good prognosis compared to the patients with lower CD90. Although the expression of CD90 in human ovarian cancer SKOV3 cells enhances the cell proliferation by MTT and anchorage-dependent growth assay, CD90 inhibits the anchorage-independent growth ability in vitro and tumor formation in vivo. CD90 overexpression suppresses the sphere-forming ability and ALDH activity and enhances the cell apoptosis, indicating that CD90 may reduce the cell growth by the properties of CSC and anoikis. Furthermore, CD90 reduces the expression of other CSC markers, including CD133 and CD24. The inhibition of CD133 is attenuated by the mutant CD90, which is replaced with RLE domain into RLD domain. Importantly, the CD90-regulated inhibition of CD133 expression, anchorage-independent growth and signal transduction of mTOR and AMPK are restored by the ß3 integrin shRNA. Our results provide evidence that CD90 mediates the antitumor formation by interacting with ß3 integrin, which provides new insight that can potentially be applied in the development of therapeutic strategies in ovarian cancer.

Differential Expression Pattern of THBS1 and THBS2 in Lung Cancer: Clinical Outcome and a Systematic-Analysis of Microarray Databases.

Thrombospondin 1 and thrombospondin 2 (THBS1 and THBS2) share similar multifunctional domains, and are known to be antiangiogenic. However, the expression pattern of THBS1 and THBS2 is different, and the specific role of THBS2 in different subtypes of lung cancer remains largely unclear. To evaluate the significance of THBS1 and THBS2 in the development of lung cancer, the present study performed a microarray-based systematic-analysis to determine the transcript levels of thrombospondins and their relation to the prognosis in lung cancer. THBS1 was in general underexpressed in lung cancer; in contrast, mRNA levels of THBS2 were markedly overexpressed in a number of datasets of non-small cell lung carcinoma (NSCLC), including lung adenocarcinoma (AC) and squamous cell carcinoma. Similar expression pattern of THBS1 and THBS2 was verified in pulmonary AC cell lines with real-time PCR analysis. The survival of lung AC patients with high THBS2 mRNA expression levels was poorer than patients with low levels of expression of THBS2. In a microarray-based analysis, genes coexpressed with THBS1 or THBS2 were determined. Pulmonary AC patients with a high expression level of sevenTSHB1-coexpressed genes (CCL5, CDH11, FYB, GZMK, LA-DQA1, PDE4DIP, and SELL) had better survival rates than those with a low expression level. Patients with a high expression of seven TSHB2-coexpressed genes (CHI3L1, COL5A2, COL11A1, FAP, MXRA5, THY1, and VCAN) had poor survival rates. Downregulation of VCAN and THBS2 with shRNA inhibited the cell proliferation in the A549 cell line. In summary, THBS1 functions as a tumor suppressor in lung adenocarcinoma. However, THBS2 may play a double-edged role in the progression of lung AC, i.e. anti-angiogenic and oncogenic function. Further study on the mechanism underlying the activity of THBS2 is warranted to have further implications for cancer diagnosis and treatment of pulmonary AC.

Systematic Analysis of Gene Expression Alterations and Clinical Outcomes for Long-Chain Acyl-Coenzyme A Synthetase Family in Cancer.

Dysregulated lipid metabolism contributes to cancer progression. Our previous study indicates that long-chain fatty acyl-Co A synthetase (ACSL) 3 is essential for lipid upregulation induced by endoplasmic reticulum stress. In this report, we aimed to identify the role of ACSL family in cancer with systematic analysis and in vitro experiment. We explored the ACSL expression using Oncomine database to determine the gene alteration during carcinogenesis and identified the association between ACSL expression and the survival of cancer patient using PrognoScan database. ACSL1 may play a potential oncogenic role in colorectal and breast cancer and play a potential tumor suppressor role in lung cancer. Co-expression analysis revealed that ACSL1 was coexpressed with MYBPH, PTPRE, PFKFB3, SOCS3 in colon cancer and with LRRFIP1, TSC22D1 in lung cancer. In accordance with PrognoScan analysis, downregulation of ACSL1 in colon and breast cancer cell line inhibited proliferation, migration, and anchorage-independent growth. In contrast, increase of oncogenic property was observed in lung cancer cell line by attenuating ACSL1. High ACSL3 expression predicted a better prognosis in ovarian cancer; in contrast, high ACSL3 predicted a worse prognosis in melanoma. ACSL3 was coexpressed with SNUPN, TRIP13, and SEMA5A in melanoma. High expression of ACSL4 predicted a worse prognosis in colorectal cancer, but predicted better prognosis in breast, brain and lung cancer. ACSL4 was coexpressed with SERPIN2, HNRNPCL1, ITIH2, PROCR, LRRFIP1. High expression of ACSL5 predicted good prognosis in breast, ovarian, and lung cancers. ACSL5 was coexpressed with TMEM140, TAPBPL, BIRC3, PTPRE, and SERPINB1. Low ACSL6 predicted a worse prognosis in acute myeloid leukemia. ACSL6 was coexpressed with SOX6 and DARC. Altogether, different members of ACSLs are implicated in diverse types of cancer development. ACSL-coexpressed molecules may be used to further investigate the role of ACSL family in individual type of cancers.

Therapeutics targeting CD90-integrin-AMPK-CD133 signal axis in liver cancer.

CD90 is used as a marker for cancer stem cell in liver cancer. We aimed to study the mechanism by which CD90 promoted liver cancer progression and identify the new therapeutic targets on CD90 signal pathway. Ectopic expression of CD90 in liver cancer cell lines enhanced anchorage-independent growth and tumor progression. Furthermore, CD90 promoted sphere formation in vitro and upregulated the expression of the cancer stem cell marker CD133. The CD133 expression was higher in CD45-CD90+ cells in liver cancer specimen. The natural carcinogenic molecules TGF-ß-1, HGF, and hepatitis B surface antigen increased the expression of CD90 and CD133. Inhibition of CD90 by either shRNA or antibody attenuated the induction of CD133 and anchorage-independent growth. Lentiviral delivery of CD133 shRNA abolished the tumorigenicity induced by CD90. Ectopic expression of CD90 induced mTOR phosphorylation and AMPK dephosphorylation. Mutation of integrin binding-RLD domain in CD90 attenuated the induction of CD133 and anchorage-independent growth. Similar results were observed after silencing ß3 integrin. Signaling analyses revealed that AMPK/mTOR and ß3 integrin were required for the induction of CD133 and tumor formation by CD90. Importantly, the energy restriction mimetic agent OSU-CG5 reduced the CD90 population in fresh liver tumor sample and repressed the tumor growth. In contrast, sorafenib did not decrease the CD90+ population. In conclusion, the signal axis of CD90-integrin-mTOR/AMPK-CD133 is critical for promoting liver carcinogenesis. Molecules inhibiting the signal axis, including OSU-CG5 and other inhibitors, may serve as potential novel cancer therapeutic targets in liver cancer.

Argininosuccinate synthetase 1 suppression and arginine restriction inhibit cell migration in gastric cancer cell lines.

Gastric cancer metastasis remains a major cause of cancer-related deaths. There is an urgent need to develop new therapeutic approaches targeting metastatic gastric cancer. Argininosuccinate synthetase 1 (ASS1) expression is increased in gastric cancer. We detected the protein expression of ASS1 in human gastric cancer cell lines (AGS, NCI-N87, and MKN45) and in murine gastric cancer cell lines (3I and 3IB2). We used vector-mediated short hairpin RNA (shRNA) expression to silence ASS1 expression in the MKN45 and 3IB2 cell lines, and analyzed the effects of this protein on cell migration and metastasis. We demonstrated that ASS1 silencing suppressed cell migration in the MKN45 and 3IB2 cell lines. ASS1 knockdown significantly reduced liver metastasis in mice after the intrasplenic implantation of 3IB2 cancer cell clones. To determine whether arginine restriction may represent a therapeutic approach to treat gastric cancer, the sensitivity of tumor cells to arginine depletion was determined in gastric cancer cells. Arginine depletion significantly inhibited cell migration in the gastric cancer cell line. The silencing of ASS1 expression in MKN45 and 3IB2 gastric cancer cells markedly decreased STAT3 protein expression. In conclusion, our results indicate that the ASS1 protein is required for cell migration in gastric cancer cell lines.

Suppression of mucin 2 promotes interleukin-6 secretion and tumor growth in an orthotopic immune-competent colon cancer animal model.

Mucin 2 (MUC2) is the major secreted mucin of the large intestine and is expressed by adenomas and mucinous carcinomas. Since colon cancer is associated with a proinflammatory microenvironment and dysregulated MUC2 expression, the aim of this study was to characterize the effects of MUC2 gene expression in colon tumor progression using colonic cancer cells. CT26 colon cancer cells were stably transfected with MUC2 siRNA (MUC2 RNAi) or a control construct containing a nonspecific sequence (scrambled RNAi). Expression of MUC2 was significantly decreased in the MUC2 RNAi cell clones. Although MUC2 suppression did not affect the cell growth of colon cancer cells in vitro, MUC2 knockdown promoted tumor growth in an orthotopic colon cancer model in vivo. MUC2 silencing also increased interleukin (IL)-6 secretion by colon cancer cells. IL-6 neutralization attenuated tumor formation by MUC2 RNAi cells; it also increased CD8 T cell infiltration into the peritoneum. Taken together, to the best of our knowledge, this is the first study indicating that the immune response to cancer cells plays an important role in tumor growth regulated by MUC2. Furthermore, given the effects of MUC2 on IL-6 secretion, its targeting may represent a potentially useful strategy to treat colonic carcinomas.

A novel cancer therapeutic using thrombospondin 1 in dendritic cells.

Induction of thrombospondin 1 (TSP-1) is generally assumed to suppress tumor growth through inhibiting angiogenesis; however, it is less clear how TSP-1 in dendritic cells (DCs) influences tumor progression. We investigated tumor growth and immune mechanism by downregulation of TSP-1 in dendritic cells. Administration of TSP-1 small hairpin RNA (shRNA) through the skin produced anticancer therapeutic effects. Tumor-infiltrating CD4(+) and CD8(+) T cells were increased after the administration of TSP-1 shRNA. The expression of interleukin-12 and interferon-γ in the lymph nodes was enhanced by injection of TSP-1 shRNA. Lymphocytes from the mice injected with TSP-1 shRNA selectively killed the tumor cells, and the cytotoxicity of lymphocytes was abolished by depletion of CD8(+) T cells. Injection of CD11c(+) TSP-1-knockout (TSP-1-KO) bone marrow-derived DCs (BMDCs) delayed tumor growth in tumor-bearing mice. Similarly, antitumor activity induced by TSP-1-KO BMDCs was abrogated by depletion of CD8(+) T cells. In contrast, the administration of shRNAs targeting TSP-2, another TSP family member, did not extend the survival of tumor-bearing mice. Finally, TSP-1 shRNA functioned as an immunotherapeutic adjuvant to augment the therapeutic efficacy of Neu DNA vaccination. Collectively, the downregulation of TSP-1 in DCs produces an effective antitumor response that is opposite to the protumor effects by silencing of TSP-1 within tumor cells.

Skin delivery of short hairpin RNA of indoleamine 2,3 dioxygenase induces antitumor immunity against orthotopic and metastatic liver cancer.

Liver cancer is one of the most malignant cancers in the world and has a high rate of metastasis. Therefore, development of a novel therapy for liver cancer is a critical issue. Indoleamine 2,3-dioxygenase (IDO) is known as a negative immune regulator in dendritic cells. Our previous study demonstrated that skin delivery of IDO short hairpin RNA (shRNA) induced antitumor immunity in subcutaneous bladder and colon tumor models. Because the immunological environment is quite different between skin and liver, it is essential to evaluate whether skin delivery of IDO shRNA is an effective treatment in metastatic and orthotopic animal tumor models. In the present study, IDO shRNA inhibited tumor growth in subcutaneous, metastatic and orthotopic liver tumor models. The cytotoxicity of splenocytes was significantly elevated in mice treated with IDO shRNA in the orthotopic and metastatic tumor models. Interleukin (IL)-12 and interferon (IFN)-gamma mRNA expression were upregulated while IL-10 was downregulated in the inguinal lymph nodes, which were collected from IDO shRNA-treated mice. Similar results were observed in the spleens of mice inoculated with IDO shRNA by gene gun. The results indicate that skin administration of IDO shRNA is an effective therapy in orthotopic and metastatic liver cancer animal models. Indoleamine 2,3-dioxygenase shRNA might be a potential new treatment for liver cancer in the future.

A combination of the metabolic enzyme inhibitor APO866 and the immune adjuvant L-1-methyl tryptophan induces additive antitumor activity.

Many types of malignant cells have a higher nicotinamide adenine dinucleotide (NAD) turnover rate than normal cells, as well as the ability to escape immune responses. Indoleamine 2,3-dioxygenase (IDO) is reported to be a negative immune regulator. Overexpression of IDO in dendritic cells is observed in tumor-draining lymph nodes. IDO-expressing dendritic cells suppress T-cell activation and promote immune tolerance. The nicotinamide phosphoribosyl transferase (NAMPT) inhibitor APO866 (also called FK866 or WK175) selectively inhibits tumor growth through intracellular NAD depletion. The IDO-specific inhibitor L-1-methyl-tryptophan (L-1MT) activates immune responses and reduces tumor volume in murine tumor models. We combined L-1MT and APO866 treatments and tested their antitumor effects in the murine gastric and bladder tumor models. In immune-competent mice, a combination of APO866 and L-1MT had a better therapeutic effect than did either L-1MT or APO866 alone. The intracellular level of NAD was suppressed by APO866 but not L-1MT. However, an additive inhibitory effect on tumor growth was not observed in tumor-bearing immune-deficient mice. The new strategy of combining a metabolic inhibitor and an immune adjuvant induced a potent therapeutic effect.