Crocetin induces cytotoxicity in colon cancer cells via p53-independent mechanisms.

OBJECTIVE: Crocin has been proposed as a promising candidate for cancer chemoprevention. The purpose of this investigation was to investigate the chemopreventive action and the possible mechanisms of crocin against human colon cancer cells in vitro. METHODS: Cell proliferation was examined using MTT assay and the cell cycle distribution fractions were analyzed using flow cytometric analysis after propidium iodide staining. Apoptosis was detected using the TUNEL Apoptosis Detection Kit with laser scanning confocal microscope. DNA damage was assessed using the alkaline single-cell gel electrophoresis assay, while expression levels of p53, cdk2, cyclin A and P21 were examined by Western blot analysis. RESULTS: Treatment of SW480 cells with crocetin (0.2, 0.4, 0.8 mmol/L) for 48 h significantly inhibited their proliferation in a concentration-dependent manner. Crocetin (0.8 mmol/L) significantly induced cell cycle arrest through p53-independent mechanisms accompanied by P21 induction. Crocetin (0.8 mmol/L) caused cytotoxicity in the SW480 cells by enhancing apoptosis and decreasing DNA repair capacity in a time-dependent manner. CONCLUSIONS: This report provides evidence that crocetin is a potential anticancer agent, which may be used as a chemotherapeutic drug.

Role of store-operated calcium entry in imperatorin-induced vasodilatation of rat small mesenteric artery.

Store-operated Ca(2+) entry (SOCE) has recently been proposed to contribute to Ca(2+) influx in vascular smooth muscle cells (VSMCs). Imperatorin is known for its potent vasodilatory effects as a dietary furanocoumarin. The study was designed to examine the hypothesis that SOCE have a functional role in imperatorin-induced vasodilation. Small mesenteric resistance arteries and mesenteric VSMCs were obtained from rats. Isometric tensions of isolated artery rings were measured by a sensitive myograph system. Laser scanning confocal microscopy was used to determine the intracellular Ca(2+) concentration of fluo-3-loaded VSMCs. Imperatorin (1-100 µM) relaxed artery rings precontracted by phenylephrine in a concentration-dependent manner. In cultured mesenteric VSMCs, passive store depletion by thapsigargin and active store depletion by phenylephrine both induced Ca(2+) influx due to SOCE. Imperatorin didn't inhibit SOCE-mediated increases in cytosolic Ca(2+) levels evoked by the emptying of the stores. In isolated artery rings, imperatorin didn't inhibit SOCE-induced contractions due to store depletion. Our results exclude SOCE mechanism of vasodilatation by imperatorin. But imperatorin is partly similar with nifedipine in vasorelaxation effect.

[Green fluorescent protein as a tracer of bone marrow stromal cells in bone tissue engineering in rhesus].

OBJECTIVE: To observe the role of green fluorescent protein (GFP) in tracing rhesus bone marrow stromal cells (rBMSCs) during tissue-engineered bone formation in vivo. METHODS: Ad5.CMV-GFP was amplified by infecting QBI-293A cells, and the bone marrow was harvested from the ilium of adult male rhesus to obtain rBMSCs, which were cultured and passaged in vitro. GFP was transfected into the third-passage rBMSCs via adenovirus vector and the labeled cells were inoculated into absorbable HA scaffold and cultured for 3 days, with untransfected rBMSCs as control, before the cell-matrix compounds were implanted into the latissimus dorsi muscles of rhesus. Samples were harvested at 6 week and embedded in paraform, and ground sections of the bone tissue were prepared to observe green fluorescence under laser scanning confocal microscope. Propidium iodide staining of the sections was also performed for observation. RESULTS: The rBMSCs grew well after GFP transfection, and green fluorescence could be seen 24 h after the transfection and became stronger till 48 h, with a positive transfection rate beyond 80%. Six weeks after cell implantation, the rBMSCs labeled by GFP-emitted green fluorescence were detected in the bone tissue under laser scanning confocal microscope. CONCLUSION: GFP can effectively trace BMSCs during bone tissue engineering, and the transplanted BMSCs constitute the main source of bone-forming cells in bone tissue engineering.

[Optical, scanning electron and atomic force microscopic observation of the microstructure of human humeral bone].

OBJECTIVE: To observe the microstructure of human long bone with scanning electron and atomic force microscopes to understand the ultrastructural organization and the composition and the morphology of the bone collagen and minerals. METHOD: Fresh human cadaveric humeral bone were fixed and dehydrated to prepare the ground sections and thin slices, respectively, which were observed for bone microstructure using optical, scanning electron and atomic force microscopy, respectively. RESULTS: Under optical microscope, the bone lacunae were in circular permutation around the Haversian canal, and the canaliculi communicated the lacunae and Haversian canal or between the lacunae. Atomic force microscopy represented distinct morphology of the large clusters of collagen, and the collagen fibers thickened in the mineralized zone and appeared in the shape of overlapping discs, whereas the calcium-phosphorus crystal displayed ellipse cylinders. The size, shape and relation of the canaliculi and lacunae were observed clearly. The bone matrix was observed in circular arrangement along the Haversian canal and the calcium-phosphorus crystal was in regular alignment under scanning electron microscope. CONCLUSION: Atomic force microscopy can help analyze the three-dimensional configuration of calcium-phosphorus crystal and bone collagen as well as the canaliculi and lacuna. The canaliculi serve as the channels mediating alimentation and molecular signals into the lacuna.

[In vitro biocompatibility of novel absorbable hydroxyapatite and AO artificial bone beta-tricalcium phosphate with rhesus bone marrow stromal cells].

OBJECTIVE: To study the in vitro biocompatibility of novel hydroxyapatite (HA) and AO artificial bone beta-tricalcium phosphate (beta-TCP) with rhesus bone marrow stromal cells (rBMSCs) . METHODS: The third passage of rBMSCs were cultured with HA and beta-TCP respectively, with the cells cultured without the materials as the control. The morphology and proliferation of cells were observed by inverted phase-contrast microscope and scanning electron microscope (SEM). MTT assay was used to semiquantitatively evaluate the cell proliferation. RESULTS: The rBMSC cocultured with HA exhibited good growth as observed under inverted phase-contrast microscope, without significant difference from the cells in the control group. Some small particles were seen pealing off from beta-TCP, and some of the cells died. Under SEM, rBMSCs showed good adhesion to HA with obvious proliferation, but the ratio of adhesive cells was not as high as that in beta-TCP group. MTT assay showed no significant difference in the cell number between HA and the control groups, but the cell number in beta-TCP group was notably less than that of control group. CONCLUSION: Novel HA has good biocompatibility with rBMSCs for bone tissue engineering, and AO artificial bone still needs improvement to serve as scaffold material for BMSCs.

Effect of pretreatment with adenosine, diazoxide or ischemic preconditioning on ischemia- reperfusion injury in the limbs of rats.

OBJECTIVE: To study the effect of ischemic preconditioning (PC) and pharmacological preconditioning with adenosine or diazoxide on ischemia-reperfusion (IR) injury in the limbs of rats. METHODS: According to different treatment received before ischemic-reperfusion injury, 66 SD rats were divided into 6 groups including a normal control and a ischemia-reperfusion control group, IP10 group in which the rats received 10-min ischemia followed by 10-min interval for reperfusion for 3 times before IR, IP5 group in which the rats were subjected to 5-min ischemia with 5-min reperfusion intervals for 3 times before IR, adenosine (Ade) pretreatment group and diazoxide (Dia) pretreatment group. Except the normal control group, which consisted of 6 rats, each group contained 12 rats, and IR injury was induced by blocking the blood flow in bilateral limbs for 4 h, followed by reperfusion for 2 or 24 h when twitch and spastic contractility of the tibialis anterior muscle and serum creatine phosphokinase (CPK) were measured. RESULTS: In IP5 and Ade pretreatment group, the twitch tension of the tibialis anterior muscle of the rats was significantly enhanced after 2 and 24 h of reperfusion, achieving the level of the normal control. The twitch tension was also enhanced in rats of IP10 group at 24 h, but at 2 h, though with less effectiveness than that in IP5 and Ade group. Dia pretreatment reduced twitch and tetanic contraction forces of the tibialis anterior muscle after 2 h of reperfusion, but obviously improved twitch tension at 24 h. Improvement in the tetanic tension, however, was seen in none of the groups. Serum CPK of IP5, IP10 and Ade groups after 2 and 24 h while Dia at only 24 h of reperfusion was obviously lower than that of IR group. CONCLUSIONS: Ischemic and Ade preconditioning can protect the limbs of rats from ischemia-reperfusion injury, and Dia has delayed protective effect. IP5 is superior than IP10 and Ade PC can produce similar effect to that of ischemic PC.