Frondanol-A5 from cucumaria frondosa induces cell cycle arrest and apoptosis in pancreatic cancer cells

Vol. 199, No. 3S, September 2004

(IFN-gamma⫺/⫺). Pmel-1 and Pmel-1IFN-gamma⫺/⫺ cells were analyzed in vitro. In vivo function was assessed by adoptive transfer of Pmel-1 or Pmel-1IFN-gamma⫺/⫺ cells into wild-type C57BL/6 or IFN-gamma⫺/⫺ mice bearing large, established subcutaneous B16 melanoma (⬎ 50mm2). Cell transfer was undertaken in combination with active immunization with fowlpox virus encoding human gp100 (rFPVhgp100) plus interleukin-2 (IL-2). Tumor size (rank-sum test) and survival (Kaplan-Meier) were analyzed. RESULTS: Pmel-1IFN-gamma⫺/⫺ cells did not produce IFNgamma, in vitro; yet, Pmel-1 and Pmel-1IFN-gamma⫺/⫺ cells were able to produce GM-CSF, TNF-alpha, MIP-1alpha, and RANTES. Adoptive transfer of Pmel-1⫹rFPVhgp100⫹IL-2 led to the regression of large tumor burden in both wild-type and IFN-gamma⫺/⫺ hosts. Pmel-1IFN-gamma⫺/⫺ T-lymphocytes were less efficacious in a wildtype host. However, when Pmel-1IFN-gamma⫺/⫺ cells were employed into IFN-gamma⫺/⫺ hosts, therapeutic efficacy was abrogated with tumor growth similar to no treatment controls. CONCLUSIONS: These data suggest that IFN-gamma is essential for treatment of large, established melanoma, but this cytokine is not necessarily produced solely by adoptively transferred antigen specific T-lymphocytes and may be provided by the host’s cells. These findings have important implications for the design of immunotherapy trials in humans.

Frondanol-A5 from cucumaria frondosa induces cell cycle arrest and apoptosis in pancreatic cancer cells Alexandra B Roginsky MD, Xian-Zhong Ding MD, PhD, Brahmchetna Singh MS, Michael Ujiki MD, M Reza Salabat MD, Chung-Yip Chan MD, Richard H Bell, Jr., MD, FACS, Peter Collin, Thomas E Adrian PhD Northwestern University, Medical College of Wisc., Coastside Bio Resources Chicago, IL INTRODUCTION: Pancreatic cancer poses a grave prognosis due to the advanced and inoperable stage at which it is usually diagnosed and lack of effective adjuvant therapy. We previously reported that a commercial product, Frondanol(r)-A5, produced from an edible sea cucumber inhibits growth of pancreatic cancer. In the present study, we examined its effects on cell cycle and apoptosis. METHODS: Cells were cultured in serum-free conditions 24 hours prior to treatment. Cell cycle and apoptosis-related proteins were evaluated by western blotting. Cells were stained with propidium iodide for cell cycle analysis. Messenger RNA was quantified by real-time PCR. RESULTS: Frondanol(r)-A5 inhibited proliferation of pancreatic cancer cells by arrest in the G2/M phase of the cell cycle and induction of apoptosis. Thymidine incorporation was concentration-dependently decreased by Frondanol(r)-A5 and was markedly inhibited in AsPC-1 (P ⬍ 0.005) and MiaPaCa-2 (P ⬍ 0.01), at 16 and 62 mcg/ml, respectively. Western blots explained the G2/M phase arrest with a decrease in cyclin A,

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cyclin B and CDC25c in treated cells versus controls at 24 hours. Frondanol(r)-A5 increased the apoptotic p21waf1 mRNA by 32fold in AsPC-1 and S2013 cells at three hours. The increase in p21waf1 protein was dramatic at three hours and persisted beyond 24 hours. Marked cleavage of caspase 3 and PARP at 24 hours and increasing by 48 hours of treatment confirmed the induction of apoptosis. CONCLUSIONS: The consistent induction cell cycle arrest and apoptosis with lack of known toxicity make Frondanol(r)-A5 a promising agent for future preventive and/or adjuvant therapy of pancreatic cancer.

Humoral and cellular immune responses in stage III-IV melanoma patients: implications for immunotherapy M Adamina MD, M Bolli MD, P Zajac PhD, WR Marti MD, FACS, D Oertli MD, FACS, GC Spagnoli MD, M Heberer MD, FACS Institute for Surgical Research and Hospital Mnagement Basel, Switzerland INTRODUCTION: To assess humoral and cytotoxic T lymphocyte responsiveness in melanoma patients undergoing immunotherapy trials. METHODS: 24 stage III (n ⫽ 5) and stage IV (n ⫽ 19) melanoma patients underwent immunization with a recombinant vaccinia virus encoding 3 tumor associated epitopes (TAA: gp100280-288, Mart127-35, tyrosinase1-9) and CD80/CD86 costimulatory molecules (rVV). Immunization occured i.d. in a first trial (17 patients) and intranodally in a second, ongoing trial (7 patients). Frequencies of CTL precursor (CTLp) specific for TAA or influenza matrix (IM) control epitope were quantified. Humoral response against rVV was measured by ELISA. RESULTS: Depending on specific responses to immunization patients were ranked as good responders (responsive to 3 or 2 epitopes) or poor responders (unresponsive or responsive to 1 epitope). All stage III patients showed CTL responses against all 3 epitopes. In contrast (p ⬍ 0.05), 7/13 stage IV patients were poor responders. 6/13 stage IV patients were good responders, one showing a complete, long lasting clinical response. Remarkably, this patient displayed also high CTLp specific for IM. rVV specific humoral response was increased (OD ⬎ 50% of pre-treatment) in all stage III patients, but only in 6/13 stage IV patients (p ⬍ 0.05). A significant correlation (p ⬍ 0.05) emerged between low rVV humoral response (OD ⬍ 50% of pre-treatment) and poor CTL responses. CONCLUSIONS: Immunocompromission in stage IV melanoma patients might hamper the induction of tumor specific CTL responses. Conversely, induction of rVV specific humoral responses doesn’t prevent the generation of CTL specific for TAA. These data suggest that stage III patients and immunocompetent stage IV patients are more likely to respond to immunotherapy.