Effect of CKBM on prostate cancer cell growth in vitro and in vivo.

Prostate carcinoma and metastasis are common among male subjects worldwide. CKBM is a drug product targeting prostate cancer in multiple ways. Prostate cancer cell lines PC3 and DU145 were treated with CKBM. The effect of CKBM on the cell's viability, cell cycle, adhesive and invasive properties and its growth in an animal model were assessed. Results indicated that CKBM inhibited PC3 and DU145 cell growth in vitro at IC(50 )values 3.923 and 4.697% respectively, and it brought about cell cycle arrest at G2/M phase. CKBM also attenuated DU145 cells to invade and adhere to extracellular matrices including Matrigel, laminin, fibronectin and collagen IV. Moreover, PC3 tumor xenograft growth was inhibited by over 60% after 28-day of 0.2, 0.4 or 0.8 ml/day CKBM treatment. The present study indicates that CKBM is effective against prostate cancer cell growth in vitro and in vivo. Further studies are required to elucidate its mechanism of action.

Plant-derived MINA-05 inhibits human prostate cancer proliferation in vitro and lymph node spread in vivo.

Few treatment options exist for metastatic prostate cancer (PC) that becomes hormone refractory (HRPC). In vitro, plant-derived MINA-05 caused dose-dependent decreases in cell numbers in HRPC cell lines LNCaP-C4-2B and PC-3, and in androgen-sensitive LNCaP-FGC, DuCaP, and LAPC-4, by WST-1 assay. MINA-05 pretreatment significantly decreased clonogenic survival in agar and on plastic at 1 x and 2 x IC50 for PC-3 (P < .05 and P < .001, respectively), and at 1/2 x, 1 x, and 2 x IC50 for LNCaP-FGC cells (P < .001). MINA-05 also induced G2M arrest of LNCaP-FGC and PC-3 cells (by flow cytometry) and caused some apoptosis in LNCaP-FGC (sub-G1 peak on flow, expression of activated caspase-3) but not in PC-3 cells. Western blotting indicated that these cell cycle changes were associated with decreased levels of regulatory proteins cyclin B1 and cdc25C. MINA-05 given daily by gavage for 39 days did not diminish primary orthotopic PC-3 growth in nude mice, but decreased the extent of lymph node invasion at higher doses. We conclude that MINA-05 induces G2M arrest, inhibits cell growth, reduces PC cell regrowth in vitro, and reduces lymph node invasion after orthotopic PC-3 cell implantation in vivo. It has potential as an adjuvant treatment for patients with PC.

Antiproliferative effects of melatonin on the growth of rat pituitary prolactin-secreting tumor cells in vitro.

Earlier studies showed that melatonin reduced the growth of 17-beta-estradiol (E(2))-induced rat pituitary prolactin-secreting tumor (prolactinoma) in vivo. The mechanisms of melatonin's inhibitory action on the prolactin-secreting tumor were further explored by investigating the in vitro effects of melatonin on the growth of pituitary prolactin-secreting tumor cells. Primary cultured prolactinoma cells from E(2)-induced rat pituitary prolactin-secreting tumor were treated with 10(-5), 10(-4) or 10(-3) m melatonin for 5 days. Apoptosis was evaluated using flow cytometry and the TdT-mediated dUTP nick-end labeling (TUNEL) method. In addition, cell viability was analyzed by (3,4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. It was found that incubation of prolactinoma cells with 10(-5), 10(-4) or 10(-3) m melatonin for 5 days inhibited cell growth and increased cell apoptosis. Furthermore, melatonin increased caspase-3 activity, Bax mRNA expression, and cytochrome c protein expression. Conversely, Bcl-2 mRNA expression and mitochondrial membrane potential were inhibited by melatonin treatment. Our results further suggest that melatonin inhibits tumor growth by inducing apoptosis of rat pituitary prolactin-secreting tumor directly via the damage of mitochondria.

Inhibitory effects of melatonin on the growth of pituitary prolactin-secreting tumor in rats.

The in vivo effects of melatonin on proliferation and apoptosis of 17-beta-estradiol (E2)-induced pituitary prolactin-secreting tumor (prolactinoma) were investigated in rats kept in 12 L/12 D (lights on: 06:00-18:00 hr). As melatonin was shown to induce apoptosis of breast and liver tumor cells, we examined whether melatonin would induce apoptosis of rat pituitary prolactinoma cells. 0.125, 0.25, 0.50 or 1.0 mg melatonin/day/rat was administrated subcutaneously at 17:30-18:00 hr. The weight of prolactinomas was measured. Apoptosis was evaluated using the TdT-mediated dUTP nick-end labeling method. It was found that treatment with 0.25 and 0.50 mg melatonin for 97 days inhibited prolactinoma cell proliferation and increased prolactinoma cell apoptosis. Furthermore, melatonin induced mRNA expression of Bax and cytochrome c protein expression. Conversely, mRNA expression of Bcl-2, and mitochondrial membrane potential were inhibited by melatonin treatment. These results suggest that melatonin inhibits the proliferation and induces apoptosis of rat pituitary prolactin-secreting tumor via perturbation of mitochondria physiology.

Suppression of primary breast, colon, gastric and bladder cancers cell growth in vitro by CKBM, a natural product.

CKBM is a product composed of natural ingredients and had been shown to possess certain anti-cancer effects in vitro and in vivo. The aim of the present study is to analyze the chemosensitivity in the treatment of primary colon, breast, gastric and bladder cancer cells by CKBM. A total of 77 patients with cancers of breast, colon, stomach or bladder were included in the present study. Primary cancer cells were isolated from the surgical removed tumors and treated with various dosages of CKBM for 5 days. ATP is then extracted and measured by luminescence assay. CKBM treatment inhibited primary colon, breast, gastric and bladder cancer growth dose-dependently. The IC values were smaller from tumor cells at early stages, when compared with the ones at later stages. The present study strongly indicated that CKBM exerted cytotoxic effect on primary cancer cells.

Immuno-regulatory effects of CKBM on the activities of mitogen-activated protein kinases and the release of cytokines in THP-1 monocytic cells.

CKBM is an herbal formula composed of five Chinese medicinal herbs (Panax ginseng, Schisandra chinensis, Fructus crataegi, Ziziphus jujube and Glycine Max) supplemented with processed Saccharomyces cerevisiae. Previous studies have demonstrated that CKBM is capable of triggering the release of IL-6 and TNFalpha from human peripheral blood mononuclear cells, and its anti-tumorigenic activity has been demonstrated in nude mice with gastric cancer. In this report, we utilized the THP-1 monocytic cell line as a cellular model to investigate how CKBM regulates the intracellular signaling of monocytes and the subsequent release of the produced cytokines. In terms of mitogen-activated protein kinase (MAPK) cascades, CKBM (20%) had no significant effect on ERK, but was linked to an inhibitory effect on JNK and a stimulatory effect on p38 MAPK. The differential responsiveness of JNK and p38 was dependent on the duration of treatment, as well as on the dosage of CKBM. Treatment of CKBM alone induced the release of IL-10 and IFNgamma, but not IL-1beta, IL-4, IL-6 and TNFbeta, while increase of intracellular Ca2+ concentration by A23187 triggered the release of IL-10 only. Interestingly, A23187 synergized with the activities of CKBM-treated THP-1 cells in terms of IL-1beta and IFNgamma production, while the IL-10 production showed no synergistic relationship between CKBM and A23187. This A23187-induced synergism was associated with a dose-dependent character towards CKBM administration. In view of the intracellular Ca2+ elevation during monocyte activation, our results suggest that CKBM can serve as a promoting agent for modulating the functions of monocytes.

Cell Cycle Arrest by a Natural Product via G2/M Checkpoint.

CKBM is a natural product that exhibits a novel anti-tumor activity through the induction of cell cycle arrest and apoptosis. We have investigated its effects on cell cycle regulation using a gastric cancer cell line, AGS. The effects of CKBM on cell proliferation, cell cycle regulation and apoptosis were analyzed using BrdU (5-bromo-2'-deoxyuridine) cell proliferation assay and flow cytometric analysis, respectively. Specific cellular protein expressions were measured using Western blot analysis. Flow cytometric analysis indicated that CKBM induced G2/M cell cycle arrest and apoptosis, whereas differential protein expressions of p21, p53 and 14-3-3sigma (stratifin) using Western blot analysis were enhanced. The differential expressions of p21, p53 and 14-3-3sigma in AGS cancer cells after CKBM treatment may play critical roles in the G2/M cell cycle arrest that blocks cell proliferation and induces apoptosis.

Anti-tumorigenic and Pro-apoptotic effects of CKBM on gastric cancer growth in nude mice.

Natural botanical products can be integrated with western medicine to optimize the treatment outcome, increase immune function and minimize the side effects from western drug treatment. CKBM is a combination of herbs and yeasts formulated based on traditional Chinese medicinal principles. Previous study has demonstrated that CKBM is capable of improving immune responsiveness through the induction of cytokine mediators, such as TNF-alpha and IL-6. In this study, we aimed to investigate the effect of this immunomodulatory drug on gastric cancer growth using a human xenograft model. Gastric cancer tissues were implanted subcutaneously into athymic nude mice followed by a 14-day or 28-day of CKBM treatment. Results showed that higher doses of CKBM (0.4 or 0.8 ml/mouse/day) produced a dose-dependent inhibitory effect on gastric tumor growth after 28-day drug treatment. This was associated with a decrease of cellular proliferation by 30% with concomitant increase in apoptosis by 97% in gastric tumor cells when compared with the control group. In contrast, CKBM showed no effect on angiogenesis in gastric tumors. This study demonstrates the anti-tumorigenic action of CKBM on gastric cancer probably via inhibition of cell proliferation and induction of apoptosis, and provides future potential targets of this drug candidate on cancer therapy.

Administration of melatonin after onset of ischemia reduces the volume of cerebral infarction in a rat middle cerebral artery occlusion stroke model.

BACKGROUND AND PURPOSE: In both permanent and transient 3-hour middle cerebral artery occlusion rat stroke models, a single intraperitoneal injection of melatonin at 5 or 15 mg/kg given before ischemia was shown to reduce infarct volume at 72 hours. The present study was conducted to examine the treatment time window when melatonin was commenced after onset of ischemia. METHODS: Adult male Sprague-Dawley rats were anesthetized to undergo right-sided middle cerebral artery occlusion for 3 hours. A single intraperitoneal injection of vehicle or melatonin at 5 mg/kg was given at 0, 1, or 3 hours after onset of ischemia. Other groups received multiple injections of vehicle or melatonin at 5 mg/kg with the first injection given at 1, 2, or 3 hours after onset of ischemia and the second and third injections at 24 and 48 hours, respectively. Multiple injections of melatonin at 15 mg/kg with the first injection given at 3 hours were also made. The infarct volume was determined at 72 hours. RESULTS: A single dose of melatonin at 5 mg/kg given at 0 or 1 but not 3 hours after onset of ischemia reduced the infarct volume. Multiple doses of melatonin at 5 mg/kg also reduced the infarct volume when the first dose was given at 1 or 2 but not 3 hours after onset. Significant hemodynamic effects were not observed. CONCLUSIONS: Our results indicate that melatonin at 5 mg/kg given as a single injection or multiple injections protects against focal cerebral ischemia when commenced within 2 hours of onset.

Melatonin potentiates phenylephrine-stimulated intracellular Ca2+ transient in smooth muscle cell of large arteries of chick embryo.

In the current study we first characterized the properties of the 2-[125I]iodomelatonin binding in pulmonary artery and aorta of chick embryo, and then determined the location of the binding site with autoradiography. Receptor binding assays were used using 2-[125I]iodomelatonin as ligand. The binding was stable, saturable, specific and reversible. Scatchard anlaysis revealed an equilibrium binding constant (Kd) of 27.12 +/- 1.34 pM and a maximum binding capacity (Bmax) of 1.93 +/- 0.19 fmol/mg protein (n = 5). Guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) increased the Kd, but reduced the Bmax, indicating the binding being coupled to a G-protein. Autoradiography revealed the binding sites in the smooth muscle layer. To delineate the physiological function of melatonin in the large artery of the chick embryo, we determined the intracellular calcium ([Ca2+]i) in smooth muscle cells of the aorta with spectrofluorometry, using fura 2-AM as calcium indicator. Melatonin at 10(-8) to 10(-5), which itself had no effect, potentiated the stimulating effect of 0.1 microM phenylephrine, a selective agonist of alpha -adrenoceptor, on [Ca2+]i. In conclusion, specific binding of 2-[125I]iodomelatonin and physiological response to melatonin suggest the existence of melatonin receptor in the smooth muscle of large arteries of the chick embryo. Melatonin potentiates the effects of alpha1-adrenergic stimulation.