Suppression of Proinflammatory and Prosurvival Biomarkers in Oral Cancer Patients Consuming a Black Raspberry Phytochemical-Rich Troche

Abstract

Black raspberries (BRB) demonstrate potent inhibition of aerodigestive tract carcinogenesis in animal models. However, translational clinical trials evaluating the ability of BRB phytochemicals to impact molecular biomarkers in the oral mucosa remain limited. The present phase 0 study addresses a fundamental question for oral cancer food-based prevention: Do BRB phytochemicals successfully reach the targeted oral tissues and reduce proinflammatory and antiapoptotic gene expression profiles? Patients with biopsy-confirmed oral squamous cell carcinomas (OSCC) administered oral troches containing freeze-dried BRB powder from the time of enrollment to the date of curative intent surgery (13.9 ± 1.27 days). Transcriptional biomarkers were evaluated in patient-matched OSCCs and noninvolved high at-risk mucosa (HARM) for BRB-associated changes. Significant expression differences between baseline OSCC and HARM tissues were confirmed using a panel of genes commonly deregulated during oral carcinogenesis. Following BRB troche administration, the expression of prosurvival genes (AURKA, BIRC5, EGFR) and proinflammatory genes (NFKB1, PTGS2) were significantly reduced. There were no BRB-associated grade 3-4 toxicities or adverse events, and 79.2% (N = 30) of patients successfully completed the study with high levels of compliance (97.2%). The BRB phytochemicals cyanidin-3-rutinoside and cyanidin-3-xylosylrutinoside were detected in all OSCC tissues analyzed, demonstrating that bioactive components were successfully reaching targeted OSCC tissues. We confirmed that hallmark antiapoptotic and proinflammatory molecular biomarkers were overexpressed in OSCCs and that their gene expression was significantly reduced following BRB troche administration. As these molecular biomarkers are fundamental to oral carcinogenesis and are modifiable, they may represent emerging biomarkers of molecular efficacy for BRB-mediated oral cancer chemoprevention.

Figure 1. Compounded BRB troches and Phase 0 clinical trial design

A, Slow release dissolvable BRB troches packaged in protective plastic container containing 30 pre-scored troches. B, Cancer patients with biopsy-confirmed OSCC were consented and enrolled into the study protocol. Participants consumed three dissolvable slow-release BRB troches q.i.d. for a cumulative daily dose of 4.3g freeze-dried BRB powder. Each patient provided oral tissue biopsies from oral tumor before and following BRB administration (OSCC1, OSCC2, respectively) and histologically “normal,” high at-risk mucosa before and following oral BRB administration (HARM1, HARM2, respectively).

Figure 1. Compounded BRB troches and Phase 0 clinical trial design

A, Slow release dissolvable BRB troches packaged in protective plastic container containing 30 pre-scored troches. B, Cancer patients with biopsy-confirmed OSCC were consented and enrolled into the study protocol. Participants consumed three dissolvable slow-release BRB troches q.i.d. for a cumulative daily dose of 4.3g freeze-dried BRB powder. Each patient provided oral tissue biopsies from oral tumor before and following BRB administration (OSCC1, OSCC2, respectively) and histologically “normal,” high at-risk mucosa before and following oral BRB administration (HARM1, HARM2, respectively).

Figure 2. Dissolution kinetics for total phenolics of BRB troches in vitro

Total polyphenolic release from BRB oral troches was estimated from λ_max 765 nm measurements using a pH6.5 phosphate buffer system over 90 minutes.

Figure 3. Analytic phytochemistry for malignant oral tissues following short-term BRB troche administration

Detection of the anthocyanins cyanidin-3-sambubioside and cyanidin-3-glucoside (peaks 1,2), cyanidin-3-xylosylrutinoside (peak 3), and cyanidin-3-rutinoside (peak 4) by LC MS/MS in representative OSCC biopsy tissues following short-term BRB troche administration. A, Patient PT035, 45y, stage 4 lateral tongue OSCC, never-smoker, 8-day BRB troche exposure. B, Patient PT036, 75y, stage 2 retromolar mucosa OSCC, current smoker, 7-day BRB exposure.

Figure 3. Analytic phytochemistry for malignant oral tissues following short-term BRB troche administration

Detection of the anthocyanins cyanidin-3-sambubioside and cyanidin-3-glucoside (peaks 1,2), cyanidin-3-xylosylrutinoside (peak 3), and cyanidin-3-rutinoside (peak 4) by LC MS/MS in representative OSCC biopsy tissues following short-term BRB troche administration. A, Patient PT035, 45y, stage 4 lateral tongue OSCC, never-smoker, 8-day BRB troche exposure. B, Patient PT036, 75y, stage 2 retromolar mucosa OSCC, current smoker, 7-day BRB exposure.

Figure 4. Patient-level transcriptional responses in OSCC biopsy tissues following short-term BRB troche administration

A, Significant reduction in gene expression levels was evident for AURKA, BIRC5, and EGFR (P < 0.05). B, The molecular biomarkers NFKB1, PTGS1, and PTGS2, which demonstrated similar transcriptional inhibition but did not reach statistical significance following Bonferroni correction, are included for comparison. x-axis, OSCC patient ID and self-reported compliance to the BRB protocol; y-axis, fold-change in gene expression.

Figure 4. Patient-level transcriptional responses in OSCC biopsy tissues following short-term BRB troche administration

A, Significant reduction in gene expression levels was evident for AURKA, BIRC5, and EGFR (P < 0.05). B, The molecular biomarkers NFKB1, PTGS1, and PTGS2, which demonstrated similar transcriptional inhibition but did not reach statistical significance following Bonferroni correction, are included for comparison. x-axis, OSCC patient ID and self-reported compliance to the BRB protocol; y-axis, fold-change in gene expression.