Modulation of the oral glucocorticoid system during black raspberry mediated oral cancer chemoprevention.

Recent reports suggest that glucocorticoids (GCs), which can be synthesized in the oral mucosa, play an important role in cancer development. Therefore, the objectives of this study were to characterize the role of the oral GC system in oral cancer, and determine the effect of black raspberry (BRB) administration on GC modulation during oral cancer chemoprevention. We determined the expression of GC enzymes in various oral cancer cell lines, and investigated the role of the GC inactivating enzyme HSD11B2 on CAL27 oral cancer cells using siRNA mediated knockdown approaches. Using two in vivo models of oral carcinogenesis with 4-nitroquinoline 1-oxide carcinogen on C57Bl/6 mice and F344 rats, we determined the effect of BRB on GC modulation during head and neck squamous cell carcinoma chemoprevention. Our results demonstrate that HSD11B2, which inactivates cortisol to cortisone, is downregulated during oral carcinogenesis in clinical and experimental models. Knockdown of HSD11B2 in oral cancer cells promotes cellular proliferation, invasion and expression of angiogenic biomarkers EGFR and VEGFA. An ethanol extract of BRB increased HSD11B2 expression on oral cancer cells. Dietary administration of 5% BRB increased Hsd11b2 gene and protein expression and reduced the active GC, corticosterone, in cancer-induced mouse tongues. Our results demonstrate that the oral GC system is modulated during oral carcinogenesis, and BRB administration upregulates Hsd11b2 during oral cancer chemoprevention. In conclusion, our findings challenge the use of synthetic GCs in head and neck cancer, and support the use of natural product alternatives that potentially modulate GC metabolism in a manner that supports oral cancer chemoprevention.

Berries vs. Disease: Revenge of the Phytochemicals.

Secondary metabolites and phytochemicals in plant-based diets are known to possess properties that inhibit the development of several diseases including a variety of cancers of the aerodigestive tract. Berries are currently of high interest to researchers due to their high dietary source of phytochemicals. Black raspberries (BRB), Rubus occidentalis, are of special interest due to their rich and diverse composition of phytochemicals. In this review, we present the most up-to-date preclinical and clinical data involving berries and their phytochemicals in the chemoprevention of a variety of cancers and diseases. BRBs possess a variety of health benefits including anti-proliferative properties, anti-inflammatory activity, activation of pro-cell-death pathways, modulation of the immune response, microbiome modulation, reduction in oxidative stress, and many more. However, little has been done in both preclinical and clinical settings on the effects of BRB administration in combination with other cancer therapies currently available for patients. With the high potential for BRBs as chemopreventive agents, there is a need to investigate their potential in combination with other treatments to improve therapeutic efficacy.

Berry Extracts and Their Bioactive Compounds Mitigate LPS and DNFB-Mediated Dendritic Cell Activation and Induction of Antigen Specific T-Cell Effector Responses.

Berries have gained widespread recognition for their abundant natural antioxidant, anti-inflammatory, and immunomodulatory properties. However, there has been limited research conducted thus far to investigate the role of the active constituents of berries in alleviating contact hypersensitivity (CHS), the most prevalent occupational dermatological disease. Our study involved an ex vivo investigation aimed at evaluating the impact of black raspberry extract (BRB-E) and various natural compounds found in berries, such as protocatechuic acid (PCA), proanthocyanidins (PANT), ellagic acid (EA), and kaempferol (KMP), on mitigating the pathogenicity of CHS. We examined the efficacy of these natural compounds on the activation of dendritic cells (DCs) triggered by 2,4-dinitrofluorobenzene (DNFB) and lipopolysaccharide (LPS). Specifically, we measured the expression of activation markers CD40, CD80, CD83, and CD86 and the production of proinflammatory cytokines, including Interleukin (IL)-12, IL-6, TNF-α, and IL-10, to gain further insights. Potential mechanisms through which these phytochemicals could alleviate CHS were also investigated by investigating the role of phospho-ERK. Subsequently, DCs were co-cultured with T-cells specific to the OVA323-339 peptide to examine the specific T-cell effector responses resulting from these interactions. Our findings demonstrated that BRB-E, PCA, PANT, and EA, but not KMP, inhibited phosphorylation of ERK in LPS-activated DCs. At higher doses, EA significantly reduced expression of all the activation markers studied in DNFB- and LPS-stimulated DCs. All compounds tested reduced the level of IL-6 in DNFB-stimulated DCs in Flt3L as well as in GM-CSF-derived DCs. However, levels of IL-12 were reduced by all the tested compounds in LPS-stimulated Flt3L-derived BMDCs. PCA, PANT, EA, and KMP inhibited the activated DC-mediated Interferon (IFN)-γ and IL-17 production by T-cells. Interestingly, PANT, EA, and KMP significantly reduced T-cell proliferation and the associated IL-2 production. Our study provides evidence for differential effects of berry extracts and natural compounds on DNFB and LPS-activated DCs revealing potential novel approaches for mitigating CHS.

Inducible TgfbR1 and Pten deletion in a model of tongue carcinogenesis and chemoprevention.

Head and neck squamous cell carcinoma (HNSCC) is a significant public health problem, with a need for novel approaches to chemoprevention and treatment. Preclinical models that recapitulate molecular alterations that occur in clinical HNSCC patients are needed to better understand molecular and immune mechanisms of HNSCC carcinogenesis, chemoprevention, and efficacy of treatment. We optimized a mouse model of tongue carcinogenesis with discrete quantifiable tumors via conditional deletion of Tgfßr1 and Pten by intralingual injection of tamoxifen. We characterized the localized immune tumor microenvironment, metastasis, systemic immune responses, associated with tongue tumor development. We further determined the efficacy of tongue cancer chemoprevention using dietary administration of black raspberries (BRB). Three Intralingual injections of 500 µg tamoxifen to transgenic K14 Cre, floxed Tgfbr1, Pten (2cKO) knockout mice resulted in tongue tumors with histological and molecular profiles, and lymph node metastasis similar to clinical HNSCC tumors. Bcl2, Bcl-xl, Egfr, Ki-67, and Mmp9, were significantly upregulated in tongue tumors compared to surrounding epithelial tissue. CD4+ and CD8 + T cells in tumor-draining lymph nodes and tumors displayed increased surface CTLA-4 expression, suggestive of impaired T-cell activation and enhanced regulatory T-cell activity. BRB administration resulted in reduced tumor growth, enhanced T-cell infiltration to the tongue tumor microenvironment and robust antitumoral CD8+ cytotoxic T-cell activity characterized by greater granzyme B and perforin expression. Our results demonstrate that intralingual injection of tamoxifen in Tgfßr1/Pten 2cKO mice results in discrete quantifiable tumors suitable for chemoprevention and therapy of experimental HNSCC.

Black raspberry extract inhibits regulatory T-cell activity in a murine model of head and neck squamous cell carcinoma chemoprevention.

Head and neck squamous cell carcinomas (HNSCC) are one of the most diagnosed malignancies globally, with a 5-year survival rate of approximately 40% to 50%. Current therapies are limited to highly invasive surgery, aggressive radiation, and chemotherapies. Recent reports have demonstrated the potential phytochemical properties of black raspberries in inhibiting the progression of various cancers including HNSCCs. However, the effects of black raspberry extracts on immune cells of the tumor microenvironment, specifically regulatory T cells during HNSCC, have not been investigated. We used a mouse model of 4-nitroquinoline-1-oxide (4NQO) chemically induced HNSCC carcinogenesis to determine these effects. C57BL/6 mice were exposed to 4NQO for 16 weeks and regular water for 8 weeks. 4NQO-exposed mice were fed the AIN-76A control mouse diet or the AIN76 diet supplemented with black raspberry extract. At terminal sacrifice, tumor burdens and immune cell recruitment and activity were analyzed in the tumor microenvironment, draining lymph nodes, and spleens. Mice fed the BRB extract-supplemented diet displayed decreased tumor burden compared to mice provided the AIN-76A control diet. Black raspberry extract administration did not affect overall T-cell populations as well as Th1, Th2, or Th17 differentiation in spleens and tumor draining lymph nodes. However, dietary black raspberry extract administration inhibited regulatory T-cell recruitment to HNSCC tumor sites. This was associated with an increased cytotoxic immune response in the tumor microenvironment characterized by increased CD8+ T cells and enhanced Granzyme B production during BRB extract-mediated HNSCC chemoprevention. Interestingly, this enhanced CD8+ T-cell antitumoral response was localized at the tumor sites but not at spleens and draining lymph nodes. Furthermore, we found decreased levels of PD-L1 expression by myeloid populations in draining lymph nodes of black raspberry-administered carcinogen-induced mice. Taken together, our findings demonstrate that black raspberry extract inhibits regulatory T-cell recruitment and promotes cytotoxic CD8 T-cell activity at tumor sites during HNSCC chemoprevention. These results demonstrate the immunomodulatory potential of black raspberry extracts and support the use of black raspberry-derived phytochemicals as a complementary approach to HNSCC chemoprevention and treatment.