The methyltransferase Setdb2 mediates virus-induced susceptibility to bacterial superinfection.

Immune responses are tightly regulated to ensure efficient pathogen clearance while avoiding tissue damage. Here we report that Setdb2 was the only protein lysine methyltransferase induced during infection with influenza virus. Setdb2 expression depended on signaling via type I interferons, and Setdb2 repressed expression of the gene encoding the neutrophil attractant CXCL1 and other genes that are targets of the transcription factor NF-κB. This coincided with occupancy by Setdb2 at the Cxcl1 promoter, which in the absence of Setdb2 displayed diminished trimethylation of histone H3 Lys9 (H3K9me3). Mice with a hypomorphic gene-trap construct of Setdb2 exhibited increased infiltration of neutrophils during sterile lung inflammation and were less sensitive to bacterial superinfection after infection with influenza virus. This suggested that a Setdb2-mediated regulatory crosstalk between the type I interferons and NF-κB pathways represents an important mechanism for virus-induced susceptibility to bacterial superinfection.

Tissue-specific production of MicroRNA-155 inhibits melanocortin 5 receptor-dependent suppressor macrophages to promote experimental autoimmune uveitis.

Tissue-specific immune regulation is an important component of the immune response relevant to many areas of immunology. The focus of this study is on tissue-specific mechanisms that contribute to autoimmune uveitis. Precise gene regulation is necessary for the proper expression of an inflammatory or regulatory response. This precision gene regulation can be accomplished by microRNA at the level of the mRNA transcript. miR-155, in particular, has a complicated role in the immune response with positive and negative inflammatory effects. In this work, we identify a decrease in miR-155 in suppressor macrophages and further examine how tissue-specific production of miR-155 impacts experimental autoimmune uveitis. Importantly, we show that eliminating miR-155 expression by the target tissue before initiation reduces disease severity, but elimination of miR-155 after the onset of inflammation does not alter the course of disease. Additionally, expression of miR-155 by the target tissue before initiation is necessary for the induction of regulatory immunity that protects from further autoimmune disease, but not after the onset of inflammation. In summary, we find a MC5r-dependent decrease in miR-155 in postexperimental autoimmune uveitis APC, miR-155 production by the target tissue is necessary for the initiation of autoimmune uveitis, and may have a role in establishing protective regulatory immunity.

Inhibition of murine hepatoma tumor growth by cryptotanshinone involves TLR7-dependent activation of macrophages and induction of adaptive antitumor immune defenses.

Cryptotanshinone (CT), a purified compound initially isolated from the dried roots of Salvia militorrhiza. Bunge, exhibits cytotoxic antitumor effects on many tumors. We have shown that CT possesses the dual capacities to concomitantly inhibit the proliferation of lung cancer cells and promote the generation of antitumor immunity. In this study, we investigated whether CT could be used to treat hepatocellular carcinoma (HCC) using a mouse Hepa1-6 model. CT inhibited the proliferation of mouse hepatoma (Hepa1-6) cells in vitro by inducing Hepa1-6 cells apoptosis through the JAK2/STAT3 signaling pathway. In addition, CT activated macrophages and polarized mouse bone marrow-derived macrophages (BMM) toward an M1 phenotype in vitro, which depended on the TLR7/MyD88/NF-κB signaling pathway. Furthermore, CT significantly inhibited the growth of syngeneic Hepa1-6 hepatoma tumors, and, in combination with anti-PD-L1 cured Hepa1-6-bearing mice with the induction of long-term anti-Hepa1-6 specific immunity. Immunoprofiling of treated Hepa1-6-bearing mice revealed that CT-promoted activation of tumor-infiltrating macrophages and dendritic cells, induction of antitumor T cell response, and infiltration of effector/memory CD8 T cells in the tumor tissue. Importantly, the immunotherapeutic effects of CT and anti-PD-L1 depended on the presence of CD8 T cells. Thus, CT and anti-PD-L1 may provide an effective immunotherapeutic regimen for human HCC based on a combination of cytotoxic effects and induction of tumor-specific immunity.

Cryptotanshinone has curative dual anti-proliferative and immunotherapeutic effects on mouse Lewis lung carcinoma.

Lung cancer is currently the leading cause of cancer-related mortality with very limited effective therapy. Screening of a variety of traditional Chinese medicines (TCMs) for their capacity to inhibit the proliferation of human lung cancer A549 cells and to induce the in vitro maturation of human DCs led to the identification of cryptotanshinone (CT), a compound purified from the TCM Salvia miltiorrhiza Bunge. Here, CT was shown to inhibit the proliferation of mouse Lewis lung carcinoma (LLC) cells by upregulating p53, downregulating cyclin B1 and Cdc2, and, consequently, inducing G2/M cell-cycle arrest of LLC cells. In addition, CT promoted maturation of mouse and human DCs with upregulation of costimulatory and MHC molecules and stimulated DCs to produce TNFα, IL-1ß, and IL-12p70, but not IL-10 in vitro. CT-induced maturation of DCs depended on MyD88 and also involved the activation of NF-κB, p38, and JNK. CT was effective in the treatment of LLC tumors and, when used in combination with low doses of anti-PD-L1, cured LLC-bearing mice with the induction of subsequent anti-LLC long-term specific immunity. CT treatment promoted T-cell infiltration and elevated the expression of genes typical of Th1 polarization in LLC tumor tissue. The therapeutic effect of CT and low doses of anti-PD-L1 was reduced by depletion of CD4 and CD8 T cells. This paper provides the first report that CT induces immunological antitumor activities and may provide a new promising antitumor immunotherapeutic.

Alarmin human α defensin HNP1 activates plasmacytoid dendritic cells by triggering NF-κB and IRF1 signaling pathways.

Human neutrophil peptide 1 (HNP1), a predominant α defensin in the azurophilic granules of human neutrophils, is an alarmin capable of inducing the migration and maturation of human myeloid/conventional dendritic cells. However, it is not determined whether it can activate plasmacytoid dendritic cells (pDCs). Herein, we found that both human pDCs and CAL-1 cells, a pDC-like cell line, produced IFNα upon treatment with HNP1. Additionally, HNP1 could promote CpG ODN-induced pDC production of proinflammatory cytokines including IFNα. HNP1 triggered activation of NF-κB and nuclear translocation of interferon regulatory factor 1 (IRF1) in CAL-1 cells. HNP1 upregulation of cytokine expression in pDCs was inhibited by blockade of NF-κB activation or knockdown of IRF1, demonstrating the importance of these two signaling events in HNP1-induced pDC activation. Using a human pDC-nude mouse model, HNP1 was shown to induce IFNα production by human pDCs in vivo. Thus, HNP1 can activate human pDCs using NF-κB and IRF signaling pathways, and HNP-induced IFN production may participate in the inflammatory pathogenesis in certain authoimmune diseases such as rheumatoid arthritis.

An antisense promoter in mouse L1 retrotransposon open reading frame-1 initiates expression of diverse fusion transcripts and limits retrotransposition.

Between 6 and 30% of human and mouse transcripts are initiated from transposable elements. However, the promoters driving such transcriptional activity are mostly unknown. We experimentally characterized an antisense (AS) promoter in mouse L1 retrotransposons for the first time, oriented antiparallel to the coding strand of L1 open reading frame-1. We found that AS transcription is mediated by RNA polymerase II. Rapid amplification of cDNA ends cloning mapped transcription start sites adjacent to the AS promoter. We identified >100 novel fusion transcripts, of which many were conserved across divergent mouse lineages, suggesting conservation of potential functions. To evaluate whether AS L1 transcription could regulate L1 retrotransposition, we replaced portions of native open reading frame-1 in donor elements by synonymously recoded sequences. The resulting L1 elements lacked AS promoter activity and retrotransposed more frequently than endogenous L1s. Overexpression of AS L1 transcripts also reduced L1 retrotransposition. This suppression of retrotransposition was largely independent of Dicer. Our experiments shed new light on how AS fusion transcripts are initiated from endogenous L1 elements across the mouse genome. Such AS transcription can contribute substantially both to natural transcriptional variation and to endogenous regulation of L1 retrotransposition.

Mouse endogenous retroviruses can trigger premature transcriptional termination at a distance.

Endogenous retrotransposons have caused extensive genomic variation within mammalian species, but the functional implications of such mobilization are mostly unknown. We mapped thousands of endogenous retrovirus (ERV) germline integrants in highly divergent, previously unsequenced mouse lineages, facilitating a comparison of gene expression in the presence or absence of local insertions. Polymorphic ERVs occur relatively infrequently in gene introns and are particularly depleted from genes involved in embryogenesis or that are highly expressed in embryonic stem cells. Their genomic distribution implies ongoing negative selection due to deleterious effects on gene expression and function. A polymorphic, intronic ERV at Slc15a2 triggers up to 49-fold increases in premature transcriptional termination and up to 39-fold reductions in full-length transcripts in adult mouse tissues, thereby disrupting protein expression and functional activity. Prematurely truncated transcripts also occur at Polr1a, Spon1, and up to ∼5% of other genes when intronic ERV polymorphisms are present. Analysis of expression quantitative trait loci (eQTLs) in recombinant BxD mouse strains demonstrated very strong genetic associations between the polymorphic ERV in cis and disrupted transcript levels. Premature polyadenylation is triggered at genomic distances up to >12.5 kb upstream of the ERV, both in cis and between alleles. The parent of origin of the ERV is associated with variable expression of nonterminated transcripts and differential DNA methylation at its 5'-long terminal repeat. This study defines an unexpectedly strong functional impact of ERVs in disrupting gene transcription at a distance and demonstrates that ongoing retrotransposition can contribute significantly to natural phenotypic diversity.

LTX-315 triggers anticancer immunity by inducing MyD88-dependent maturation of dendritic cells.

LTX-315 is a synthetic cationic oncolytic peptide with potent anticancer activity but limited toxicity for non-malignant cells. LTX-315 induces both immunogenic tumor cell death and generation of tumor-specific immune responses in multiple experimental tumor models. Given the central role of dendritic cell (DC) maturation in the induction of antigen-specific immunity, we investigated the effect of LTX-315 treatment on the maturation of tumor-infiltrating DCs (TiDCs) and the generation of anti-melanoma immunity. We found that LTX-315 treatment induces the maturation of DCs, both indirectly through the release of cancer cell-derived damage-associated molecular patterns (DAMPs)/alarmins and nucleic acids (DNA and RNA) capable of triggering distinct Toll-like receptor (TLR) signaling, and, directly by activating TLR7. The latter results in the ignition of multiple intracellular signaling pathways that promotes DC maturation, including NF-κB, mitogen activated protein kinases (MAPKs), and inflammasome signaling, as well as increased type 1 interferon production. Critically, the effects of LTX-315 on DCs the consequent promotion of anti-melanoma immunity depend on the cytosolic signal transducer myeloid differentiation response gene 88 (MyD88). These results cast light on the mechanisms by which LTX-315 induces DC maturation and hence elicits anticancer immunity, with important implications for the use of LTX-315 as an anticancer immunotherapeutic.

Alpha synuclein, the culprit in Parkinson disease, is required for normal immune function.

Alpha-synuclein (αS) is causally involved in the development of Parkinson disease (PD); however, its role in normal vertebrate physiology has remained unknown. Recent studies demonstrate that αS is induced by noroviral infection in the enteric nervous system of children and protects mice against lethal neurotropic viral infection. Additionally, αS is a potent chemotactic activator of phagocytes. In this report, using both wild-type and αS knockout mice, we show that αS is a critical mediator of inflammatory and immune responses. αS is required for the development of a normal inflammatory response to bacterial peptidoglycan introduced into the peritoneal cavity as well as antigen-specific and T cell responses following intraperitoneal immunization. Furthermore, we show that neural cells are the sources of αS required for immune competence. Our report supports the hypothesis that αS accumulates within the nervous system of PD individuals because of an inflammatory/immune response.

Inhibition of two-pore channels in antigen-presenting cells promotes the expansion of TNFR2-expressing CD4+Foxp3+ regulatory T cells.

CD4+Foxp3+ regulatory T cells (Tregs) are pivotal for the inhibition of autoimmune inflammatory responses. One way to therapeutically harness the immunosuppressive actions of Tregs is to stimulate the proliferative expansion of TNFR2-expressing CD4+Foxp3+ Tregs via transmembrane TNF (tmTNF). Here, we report that two-pore channel (TPC) inhibitors markedly enhance tmTNF expression on antigen-presenting cells. Furthermore, injection of TPC inhibitors including tetrandrine, or TPC-specific siRNAs in mice, increases the number of Tregs in a tmTNF/TNFR2-dependent manner. In a mouse colitis model, inhibition of TPCs by tetrandrine markedly attenuates colon inflammation by expansion of Tregs Mechanistically, we show that TPC inhibitors enhance tmTNF levels by disrupting surface expression of TNF-α-converting enzyme by regulating vesicle trafficking. These results suggest that the therapeutic potential of TPC inhibitors is mediated by expansion of TNFR2-expressing Tregs and elucidate the basis of clinical use in the treatment of autoimmune and other inflammatory diseases.

Oral human papillomavirus infection before and after treatment for human papillomavirus 16-positive and human papillomavirus 16-negative head and neck squamous cell carcinoma.

PURPOSE: Oral human papillomavirus (HPV) infection is a risk factor for head and neck squamous cell carcinoma (HNSCC), and is a concern for patients with HPV-positive HNSCC and their partners. The prevalence of oral HPV infection before and after cancer therapy was investigated among patients with HPV16-positive and HPV16-negative HNSCC. EXPERIMENTAL DESIGN: Serial oral rinse samples (ORS) were collected from a cohort of 135 HNSCC cases as frequently as every 3 months for up to 3 years. Tumor HPV status was determined by HPV16 in situ hybridization. HPV was detected in ORS by consensus PCR and line blot hybridization. The HPV16 variants in positive oral rinse-tumor pairs were determined by sequencing. The odds of oral HPV infection among HPV16-positive and HPV16-negative cases were compared by use of generalized estimating equations. RESULTS: Patients were followed for a median of 21 months and provided a median of 4 samples. Forty-four of 135 patients had HPV16-positive tumors. HPV16-positive cases were more likely than HPV16-negative cases to have an oral HPV infection detected before (odds ratio, 8.6; 95% confidence interval, 3.5-21) and after therapy (OR, 2.9; 95% confidence interval, 1.1-7.4). Oral infections by HPV16 and other high-risk, but not low-risk, types were more common among HPV16-positive cases both before and after therapy. Most HPV16 variants in ORS were European, unique, and identical to that in the tumor. Persistence of a type-specific oral infection was demonstrable for as long as 5 years. CONCLUSION: Oral high-risk HPV infections are more frequent among patients with HPV16-positive than HPV16-negative HNSCC, consistent with a behavioral and/or biological disposition to infection.

High Glucose Promotes Human Glioblastoma Cell Growth by Increasing the Expression and Function of Chemoattractant and Growth Factor Receptors.

Diabetes mellitus, characterized by hyperglycemia, is considered as a risk factor of cancers including malignant gliomas. However, the direct effect of high glucose on cancer cell behavior is not clear. We therefore investigated the effect of hyperglycemia on the growth of human glioblastoma (GBM) cells. Our results revealed that high glucose (HG) promoted the proliferation and inhibited the apoptosis of a human GBM cell line U87. Mechanistically, HG upregulated the expression and function of a G-protein coupled chemoattractant receptor (GPCR) formyl peptide receptor 1 (FPR1) and epidermal growth factor receptor (EGFR) on GBM cells, which upon activation by their agonists, promoted cell migration and proliferation. In addition, the invasiveness and the production of VEGF by U87 cells were enhanced under HG conditions, the effects of which were mediated by FPR1 and EGFR agonists. The tumor promoting activity of HG was further substantiated by increased tumorigenicity and growth of xenograft tumors formed by GBM cells in nude mice with induced diabetes mellitus. Thus, our study demonstrates the capacity of HG to promote GBM progression via enhancement of the function of chemoattractant and growth factor receptors.

HMGN1 and R848 Synergistically Activate Dendritic Cells Using Multiple Signaling Pathways.

High mobility group nucleosome-binding protein 1 (HMGN1 or N1) is a Th1-polarizing alarmin, but alone is insufficient to induce antitumor immunity. We previously showed that combination of N1 and R848, a synthetic TLR7/8 agonist, synergistically activates dendritic cells (DCs) and induces therapeutic antitumor immunity, however, it remained unclear how N1 and R848 synergistically activate DCs. Here, we show that co-stimulation with N1 and R848 of human monocyte-derived DCs (MoDCs) markedly upregulated DC's surface expression of CD80, CD83, CD86, and HLA-DR, as well as synergistic production of pro-inflammatory cytokines including IL-12p70, IL-1ß, and TNF-α. This combination also synergistically activated NF-κB and multiple MAPKs that are involved in DC maturation. Moreover, N1 and R848 synergistically increased nuclear translocation of interferon (IFN) regulatory transcription factors (e.g., IRF3 and IRF7) and promoted the expression of type 1 IFNs such as IFN-α2, IFN-α4, and IFN-ß1. Similar signaling pathways were also induced in mouse bone marrow-derived DCs (BMDCs). RNA-seq analysis in human MoDCs revealed that N1 plus R848 synergistically upregulated the expression of genes predominantly involved in DC maturation pathway, particularly genes critical for the polarization of Th1 immune responses (e.g., IL12A, IL12B, and IFNB1, etc.). Overall, our findings show that (1) N1 synergizes with R848 in activating human and mouse DCs and (2) the synergistic effect based on various intracellular signaling events culminated in the activation of multiple transcriptional factors. These findings have important implications for future clinical trials since N1 and R848 synergistically promoted optimal Th1 lineage immune responses resulting in tumor rejection in mice.

Blockade of TNFR2 signaling enhances the immunotherapeutic effect of CpG ODN in a mouse model of colon cancer.

Through the tumor necrosis factor (TNF) receptor type II (TNFR2), TNF preferentially activates, expands, and promotes the phenotypic stability of CD4+Foxp3+ regulatory T (Treg) cells. Those Treg cells that have a high abundance of TNFR2 have the maximal immunosuppressive capacity. We investigated whether targeting TNFR2 could effectively suppress the activity of Treg cells and consequently enhance the efficacy of cancer immunotherapy. We found that, relative to a suboptimal dose of the immunostimulatory Toll-like receptor 9 ligand CpG oligodeoxynucleotide (ODN), the combination of the suboptimal dose of CpG ODN with the TNFR2-blocking antibody M861 more markedly inhibited the growth of subcutaneously grafted mouse CT26 colon tumor cells. This resulted in markedly fewer TNFR2+ Treg cells and more interferon-γ-positive (IFN-γ+) CD8+ cytotoxic T lymphocytes infiltrating the tumor and improved long-term tumor-free survival in the mouse cohort. Tumor-free mice were resistant to rechallenge by the same but not unrelated (4T1 breast cancer) cells. Treatment with the combination of TNFR2-blocking antibody and a CD25-targeted antibody also resulted in enhanced inhibition of tumor growth in a syngeneic 4T1 mouse model of breast cancer. Thus, the combination of a TNFR2 inhibitor and an immunotherapeutic stimulant may represent a more effective treatment strategy for various cancers.

Therapeutic vaccine to cure large mouse hepatocellular carcinomas.

Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide with limited therapeutic options. Here we report the development of a therapeutic vaccination regimen (shortened as 'TheraVac') consisting of intratumoral delivery of high-mobility group nucleosome-binding protein 1 (HMGN1), R848/resiquimod, and one of the checkpoint inhibitors (e.g. anti-CTLA4, anti-PD-L1, or low dose of Cytoxan). C57BL/6 mice harboring large (approximately 1 cm in diameter) established subcutaneous Hepa1-6 hepatomas were cured by intratumoral injections of TheraVac and became tumor-free long-term survivors. Importantly, the resultant tumor-free mice were resistant to re-challenge with Hepa1-6 hepatoma, not B16 melanoma, demonstrating the acquisition of hepatoma-specific immunity in the absence of any administered tumor antigen. Mechanistic studies showed that upon treatment with TheraVac, Hepa1-6-bearing mice generated increased Hepa1-6-specific CTLs in the draining lymph nodes and showed greatly upregulated expression of CXCL9, CXCL10, and IFN-γ and elevated infiltration of T lymphocytes in tumor tissues. Treatment of large Hepa1-6 hepatomas on one mouse flank also eliminated smaller (approximately 0.5 cm in diameter) hepatomas implanted on the other flank. Thus, TheraVac has potential as a curative immunotherapeutic regimen for the treatment of human HCC.

Development of a Curative Therapeutic Vaccine (TheraVac) for the Treatment of Large Established Tumors.

Harnessing immune system to treat cancer requires simultaneous generation of tumor-specific CTLs and curtailment of tumor immunosuppressive environment. Here, we developed an immunotherapeutic regimen capable of eliminating large established mouse tumors using HMGN1, a DC-activating TLR4 agonist capable of inducing anti-tumor immunity. Intratumoral delivery of HMGN1 with low dose of Cytoxan cured mice bearing small (∅ ≈ 0.5 cm), but not large (∅ ≈ 1.0 cm) CT26 tumors. Screening for activators capable of synergizing with HMGN1 in activating DC identified R848. Intratumoral delivery of HMGN1 and R848 plus Cytoxan eradicated large established CT26 tumors. The resultant tumor-free mice were resistant to subsequent challenge with CT26, indicating the generation of CT26-specific protective immunity. This immunotherapeutic regimen caused homing of tumor-infiltrating DC to draining lymph nodes and increased infiltration of T cells into tumor tissues. Cytoxan in this regimen could be replaced by anti-CTLA4) or anti-PD-L1. Importantly, this immunotherapeutic regimen was also curative for large established mouse Renca and EG7 tumors. Thus, we have developed a curative therapeutic vaccination regimen dubbed 'TheraVac' consisting of HMGN1 and R848 plus a checkpoint inhibitor, that can, without using exogenous tumor-associated antigen(s), eliminate various large tumors and induce tumor-specific immunity.

TNFR2 expression by CD4 effector T cells is required to induce full-fledged experimental colitis.

There is now compelling evidence that TNFR2 is constitutively expressed on CD4(+) Foxp3(+) regulatory T cells (Tregs) and TNF-TNFR2 interaction is critical for the activation, expansion and functional stability of Tregs. However, we showed that the expression of TNFR2 was also up-regulated on CD4(+) Foxp3(-) effector T cells (Teffs) upon TCR stimulation. In order to define the role of TNFR2 in the pathogenic CD4 T cells, we compared the effect of transferred naïve CD4 cells from WT mice and TNFR2(-/-) mice into Rag 1(-/-) recipients. Transfer of TNFR2-deficient Teff cells failed to induce full-fledged colitis, unlike WT Teffs. This was due to defective proliferative expansion of TNFR2-deficient Teff cells in the lymphopenic mice, as well as their reduced capacity to express proinflammatory Th1 cytokine on a per cell basis. In vitro, the proliferative response of TNFR2 deficient naïve CD4 cells to anti-CD3 stimulation was markedly decreased as compared with that of WT naïve CD4 cells. The hypoproliferative response of TNFR2-deficient Teff cells to TCR stimulation was associated with an increased ratio of p100/p52, providing a mechanistic basis for our findings. Therefore, this study clearly indicates that TNFR2 is important for the proliferative expansion of pathogenic Teff cells.

Enhanced immunostimulatory effects of DNA-encapsulated peptide hydrogels.

DNA that encodes tumor-specific antigens represents potential immunostimulatory agents. However, rapid enzymatic degradation and fragmentation of DNA during administration can result in limited vector expression and, consequently, poor efficacy. These challenges have necessitated the use of novel strategies for DNA delivery. Herein, we study the ability of cationic self-assembling peptide hydrogels to encapsulate plasmid DNA, and enhance its immunostimulatory potential in vivo. The effect of network charge on the gel's ability to retain the DNA was assessed employing three gel-forming peptides that vary systematically in formal charge. The peptide HLT2, having a formal charge of +5 at neutral pH, was optimal in encapsulating microgram quantities of DNA with little effect on its rheological properties, allowing its effective syringe delivery in vivo. The plasmid, DNA(TA), encapsulated within these gels encodes for a melanoma-specific gp100 antigen fused to the alarmin protein adjuvant HMGN1. Implantation of DNA(TA)-loaded HLT2 gels into mice resulted in an acute inflammatory response with the presence of polymorphonuclear cells, which was followed by infiltrating macrophages. These cellular infiltrates aid in the processing of encapsulated DNA, promoting increased lymphoproliferation and producing an enhanced immune response mediated by CD4+/IFNγ+ expressing Th1 cells, and complemented by the formation of gp100-specific antibodies.

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.