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Background: We have previously demonstrated the potent in vitro antiproliferative effects of keyhole limpet hemocyanin (KLH) against melanoma. Our prior studies directed us to hypothesize that KLH would be effective in vivo against melanoma, alone and in combination with conventional immunotherapy., Methods: Mice were inoculated with 2 x 10(7) HTB68 cells and randomized to 6 groups. Treatment groups consisted of control, KLH 200 microg, alpha interferon (AIFN) 1000 IU, interleukin-2 (IL-2) 5000 IU, KLH + AIFN, and KLH + IL-2., Results: KLH + IL-2 exhibited the greatest reduction in tumor volume (30%) as compared to control (P = .014), followed by KLH + AIFN (28%, P = .031). Singly treated animals had less tumor inhibition: IL-2 (30%, P = .022), KLH (18%, not significant), and AIFN (16%, not significant)., Conclusions: KLH augments the effects of AIFN, one of the standard immunotherapeutic agents against melanoma in vivo. Further in vivo and early clinical studies into the effects of KLH as both a single and combined agent are warranted.

Introduction: Our hypothesis was that keyhole limpet hemocyanin (KLH) would augment the effects of standard immunotherapies for melanoma including interferon-alpha (AIFN) and interleukin (IL)-2., Methods: The HTB68 melanoma cell line was treated with KLH, AIFN, and IL-2 as single and combined agents. Cell viability, apoptotic activity, and vascular endothelial growth factor levels were all evaluated., Results: Cell growth was reduced with KLH (28%), AIFN (54%), and IL-2 (29%) (all P < .001). KLH and IL-2 combined exhibited a 47% inhibition of cell growth, whereas KLH and AIFN combined yielded a 67% reduction in cell growth (both P < .001). KLH and AIFN combined significantly increased both early (10%) and late (14%) apoptotic activity compared with controls (5% and 7%, P < .001)., Conclusions: The additive effects exhibited by the combination of KLH with AIFN or IL-2 are encouraging and support combination therapy as an effective treatment for this aggressive disease.

Background: We hypothesized that keyhole limpet hemocyanin (KLH) would reduce cellular proliferation and effect apoptosis of melanoma cell lines in vitro., Methods: Two human melanoma cell lines (HTB68 and HTB72) were subjected to a dose-response treatment regimen of KLH (0.4 microg to 100 microg/well). Cell viability was tested by MTT assay (SIGMA, St Louis, MO) at 72 hours. Apoptosis and necrosis were measured by the Annexin V FITC assay (Biovision Inc, Mountain View, CA)., Results: Melanoma cell proliferation was significantly reduced in the HTB68 cell line treated with 6.3 microg or higher doses of KLH. A significant reduction in cell growth was also observed in the HTB72 cells at 50 and 100 microg of KLH. KLH increased early apoptotic activity, whereas both late apoptosis and necrosis were decreased by the addition of KLH., Conclusions: KLH significantly reduces cellular proliferation in vitro in melanoma, via early apoptotic pathways. The results warrant in vivo studies into the effects of KLH in melanoma.

Background: We have previously shown the inhibitory effects of keyhole limpet hemocyanin (KLH) against breast and pancreatic cancer in vitro. We hypothesize that its actions in breast and pancreas cancer cells are via apoptotic or cytokine pathways., Methods: Two breast cancer cell lines, ZR75-1 and MCF-7, and one pancreas cancer cell line, PANC-1, were treated with KLH at 500 mug, 250 mug, and 250 ng/mL. Cell viability, cytokine production, and apoptosis were measured., Results: Significant growth inhibition was observed in all cell lines at all KLH concentrations tested. Significant changes in cytokine production were observed in all cell lines. An increase in early and late apoptotic activity was observed in the MCF-7, whereas a reduction in late apoptotic activity was observed in the ZR75-1 cells., Conclusions: KLH directly inhibits the growth of human breast and pancreas cancer in vitro by apoptotic and nonapoptotic mechanisms.

Background: Keyhole limpet hemocyanin (KLH) is a recently described immune stimulant and hapten carrier derived from a circulating glycoprotein of the marine mollusk Megathura crenulata. We previously reported that KLH has significant antiproliferative effects in vitro against breast, pancreas, and prostate cancers. We hypothesized that KLH would be effective against Barrett's esophageal adenocarcinoma in an in vitro model., Methods: Barrett's esophageal adenocarcinoma cell lines (SEG-1 and BIC-1) were cultured using standard techniques. Cells were plated at 1 x 10(5) and KLH was added at concentrations ranging from 400 ng to 100 microg/well. After 24 and 72 h incubation, cells were assayed for viability using the MTT technique. Statistical analysis was performed using ANOVA. Apoptosis was evaluated using a cell death detection kit after 16 hours of incubation with KLH., Results: KLH treatment significantly (p < 0.001) reduced viability in a dose and time-dependent manner. Apoptosis was increased in treated SEG-1 cells, but no changes in apoptosis were seen in treated BIC-1 cells., Conclusions: KLH directly inhibits the growth of human Barrett's esophageal cancer in vitro by apoptotic and nonapoptotic mechanisms.

Background: Keyhole limpet hemocyanin (KLH) is a recently described immune stimulant and hapten carrier derived from a circulating glycoprotein of the marine mollusk Megathura crenulata. It has been reported to be a potent form of intravesical immunotherapy for the treatment of transitional cell carcinoma of the bladder and has been used in a variety of genitourinary tumors. We hypothesized that KLH would be effective against other cancer cells in vitro., Methods: Multiple cancer cell lines were tested, including estrogen-dependent breast (MCF-7), estrogen-independent breast (ZR75-1), pancreas (PANC-1, MIA-PaCa), and prostate (DU145). Serial twofold dilutions of KLH were prepared in sterile 96-well plates. Dose-response curves were performed beginning with a concentration of 100 microg of KLH/well and ending at a concentration of 0.8 ng/well. Cells were added at concentrations of 5 x 10(4) cells per well. Cell viability was evaluated at 24 and 72 h by MTT assay at an absorbance of 570 nm., Results: Significant (P < 0.05) cancer cell growth inhibition was observed in four of the five cell lines tested at both time treatment intervals. The breast cancer line ZR75-1 exhibited a mean growth inhibition of 43 +/- 1.1% (range 37 to 59%) at 72 h, whereas treated MCF-7 cells had an average of 39 +/- 9.1% growth inhibition (range 35 to 44%) at these same concentrations. Treated PANC-1 cells had a mean growth inhibition of 19 +/- 0.8% (range 4 to 46%) at 72 h. The DU145 prostate cancer cell line averaged a 6 +/- 1.3% growth inhibition (range -19 to 55%) over the concentrations tested., Conclusions: The direct growth inhibition of multiple tumor cell-lines exhibited by KLH is significant and warrants further in vitro mechanistic studies and in vivo experiments. Investigation into the efficacy and mechanism of response could directly lead to more effective treatment regimens for patients suffering from these diseases.