Thymoquinone reduces mouse colon tumor cell invasion and inhibits tumor growth in murine colon cancer models

We have shown that thymoquinone (TQ) is a potent antitumor agent in human colorectal cancer cells. In this study, we evaluated TQ's therapeutic potential in two different mice colon cancer models [1,2-dimethyl hydrazine (DMH) and xenografts]. We also examined TQ effects on the growth of C26 mouse colorectal carcinoma spheroids and assessed tumor invasion in vitro. Mice were treated with saline, TQ, DMH, or combinations once per week for 30 weeks and the multiplicity, size and distribution of aberrant crypt foci (ACF) and tumors were determined at weeks 10, 20 and 30. TQ injected intraperitoneally (i.p.) significantly reduced the numbers and sizes of ACF at week 10; ACF numbers were reduced by 86%. Tumor multiplicity was reduced at week 20 from 17.8 in the DMH group to 4.2 in mice injected with TQ. This suppression was observed at week 30 and was long-term; tumors did not re-grow even when TQ injection was discontinued for 10 weeks. In a xenograft model of HCT116 colon cancer cells, TQ significantly (P < 0.05) delayed the growth of the tumor cells. Using a matrigel artificial basement membrane invasion assay, we demonstrated that sub-cyto-toxic doses of TQ (40 microM) decreased C26 cell invasion by 50% and suppressed growth in three-dimensional spheroids. Apoptotic signs seen morphologically were increased significantly in TQ-treated spheroids. TUNEL staining of xenografts and immunostaining for caspase 3 cleavage in DMH tumors confirmed increased apoptosis in mouse tumors in response to TQ. These data should encourage further in vivo testing and support the potential use of TQ as a therapeutic agent in human colorectal cancer.     

Growth-promoting effects of gastrin on mouse colon cancer cells in vitro: Absence of autocrine effects

Summary We have previously demonstrated trophic effects of gastrin on mouse colon cancer (MC-26) cells, in vivo, and demonstrated the presence of gastrin receptors (GR) on these cells. The cellular and intracellular mechanism by which gastrin expresses trophic effects on colon cancer cells is, however, as yet unknown. For us to start investigating the possible mechanisms involved, it was important that we first develop an in vitro model, in which gastrin expresses its trophic effects directly on the MC-26 cells. The growth-promoting effects of gastrin on the MC-26 cells were examined in various in vitro culture models, in terms of [³H]thymidine incorporation and cell number. A significant trophic effect of gastrin could be demonstrated on quiescent cells in culture, in the absence of serum. The optimal cell-culture conditions for observing trophic effects of gastrin were defined and included a 24-h period of rapid growth of MC-26 cells in serum-supplemented normal growth medium, followed by a 24-h period of culture in serum-free medium containing an optimal dose (1.0 mM) of thymidine, to achieve growth-arrest of the cells. Addition of gastrin (0.5 to 25 nM) to the quiescent, growth-arrested cells resulted in significant dose-dependent increases in both the incorporation of [³H]thymidine uptake by the cells, and a significant increase in cell number. The concentration of GR on the growth-arrested quiescent MC-26 cells in culture was significantly increased compared to the GR concentration on the control, asynchronized cells. The increased presence of GR on the growth-arrested, synchronized MC-26 cells may have allowed us to observe a significant trophic effect of gastrin on the MC-26 cells, in vitro itself. To determine if gastrin was functioning as an autocrine growth factor for MC-26 cells, we examined the effect of gastrin antibodies on the growth of MC-26 cells; no significant effect of the antigastrin IgG on the growth of MC-26 cells was observed. Supported by grants from the National Institutes of Health, Bethesda, MD (PO1 DK 35608 and CA 38651, and a grant from the American Cancer Society, Washington, DC (PDT-220).     

Effects of fibroblast growth factor 2 and insulin-like growth factor II on the development of parthenogenetic mouse embryos in vitro

Most parthenogenetic embryos (PEs) in mammals die shortly after implantation, and this failure to develop is associated with genomic imprinting. We have examined the influence of human recombinant basic fibroblast growth factor 2 (FGF-2) and human recombinant insulin-like growth factor II (ICF-II) on the development of (CBA x C57BL/6)F1 parthenogenetic mouse embryos. Embryos were treated in vitro at the morula stage with different doses of FGF-2 and, after their development to blastocysts, transferred to pseudopregnant recipients. The optimal doses of FGF-2 did not affect the number of forming and implanting blastocysts, but increased, from 20 to 42%, the number of embryos developing to somite stages. PEs (18-21 somites) treated with an optimal dose of FGF-2 were explanted for further development in culture by treatment with the second growth factor, IGF-II. Eighty-three percent of those embryos cultured with IGF-II (2.5 microg/ml) developed to 35 or more somites, as compared with 36% of embryos cultured without any growth factors (P < 0.01). Also, a significantly higher proportion of PEs developed to 40-50 somites in this case. These results show that the in vitro treatment of PEs with FGF-2 at the morula stage increases the number of somite embryos, and the second treatment of somite PEs with IGF-II in culture medium prolongs their development significantly.     

Effects of the lncRNA ENST00000623984 on colon cancer and the biological characteristics of colon cancer cells

The aim of this study was to explore the effects of the lncRNA ENST00000623984 on colorectal cancer. In this study, the expression levels of ENST000000623984 were first examined in tumor tissue and adjacent normal tissue from 40 patients with colorectal cancer and LoVo cells using quantitative real-time PCR. By siRNA transfection, ENST00000623984 expression was knocked down. Using flow cytometry, cell cycle progression and cell viability were examined in basal and knockdown LoVo cells. The CCK-8 assay was used to assess the cell proliferation rate, and the Transwell assay was used to determine the migration and invasion abilities. The ENST000000623984 expression level was increased in colorectal cancer. Knockdown of ENST000000623984 reduced cell viability, proliferation rate, cell migration and invasion. These results suggested that lncRNA ENST000000623984 may be involved in colorectal cancer development.Key words: LncRNA, ENST00000623984, colorectal cancer, expression, PCR     

Photoperiod influences the growth of colon cancer in mice

We have developed a mouse colon adenocarcinoma cell line that produces tumors in a dose-dependent manner when injected subcutaneously. Our previous work has demonstrated its sequential pattern of tumor area and weight under 12L:12D (12 hours light, 12 hours darkness) photoperiod. This study investigated whether shorter (6L:18D) or longer (18L:6D) photoperiods alter tumor growth. Significantly greater tumor area, weight, and group mortality were found in mice exposed to 12L:12D photoperiods as compared to either 6L:18D or 18L:6D photoperiods, and difluoromethylornithine (DFMO) was a more effective inhibitor of tumor growth under the 6L:18D photoperiod compared to 12L:12D. These results demonstrate an important role of photoperiod on tumor growth.     

Amiloride and guggulsterone suppression of esophageal cancer cell growth in vitro and in nude mouse xenografts

Esophageal adenocarcinoma is increasing in the US and Western countries and frequent gastresophageal reflux or gastresophageal reflux disease carrying gastric acid and bile acid could contribute to esophageal adenocarcinogenesis. This study was designed to detect the expression of gastric acid-inducing gene Na＋/H＋ exchanger-1 （NHE-1） ex vivo and then to explore targeting of NHE-1 expression or activity to control esophageal cancer cell viability in vitro and in nude mouse xenografts. The data showed that NHE-1 was highly expressed in esophageal adenocarcinoma tissues （66 of 101 cases [65.3%｜, but not in normal esophageal squamous cell epithelium （1 of 26 cases [3.8~0]）. Knockdown of NHE-1 expression using NHE-1 shRNA or inhibition of NHE-1 activity using the NHE-1 inhibitor amiloride suppressed viability and induced apoptosis in esophageal cancer cells. Molecularly, amiloride inhibited expression of cyclooxygenase-2 and matrix metallopeptidase-9 but not NHE-1 mRNA in esophageal cancer cells. A combination of amiloride and guggulsterone （a natural bile acid receptor inhibitor） showed more than additive effects in suppressing esophageal cancer cell growth in vitro and in nude mouse xenografts. This study suggests that inhibition of NHE-1 expression or activity or combination of amiloride and guggulsterone could be useful in control of esophageal adenocarcinoma.     

The progression of 1,2-dimethylhydrazine-induced colon tumourigenesis stimulates growth and cell proliferation in the colon of the host mouse

Abstract. The aim of this study was to characterize tissue responses attributable to the stage of tumourigenesis in CF-1 mice. The study involved initiating colon carcinogenesis by eight weekly subcutaneous injections of 1,2-dimethylhydrazine (DMH at 12mg/kg body weight), while the control mice received injections of the carrier vehicle. All mice were killed for examination of tissue responses at 24 weeks after the completion of the injections. Each mouse was classified into one of four groups based on the histology of the most advanced colonic lesion. The four groups or stages were: 1 no lesions (non-DMH-treated mice); 2 mice with aberrant crypt foci (ACF); 3 mice with one or more adenomas (AD) and 4 mice with an adenocarcinoma (AC). Significant tissue specific responses were identified and related to the stage of tumourigenesis. For example, mice with AC demonstrated a stage specific and marked hypertrophy of the entire colon and spleen, while demonstrating loss of body weight with no change in weight of the thymus or liver. The colonic crypts from these AC-bearing mice demonstrated hyperproliferation and an upward shift of the proliferative zone, with a concurrent loss of iron, but not of calcium, copper, magnesium or zinc in the liver. Splenomegaly was attributed to transition of the tumour to an invasive state. It is proposed that the AC produces a blood-borne trophic factor which helps explain a field effect on the colonic epithelium far removed from the growing AC. This field effect can help explain how biopsy of the large bowel (usually taken from the rectum in humans) can provide morphokinetic information predictive of the stage of colon carcinogenesis.     

Prenatal alpha-difluoromethylornithine treatment: effects on postnatal renal growth and function in the rat

DFMO (alpha-difluoromethylornithine) is a specific irreversible inhibitor of ornithine decarboxylase (ODC), a key enzyme in the biosynthesis of polyamines, which in turn control macromolecule synthesis during cell proliferation. The current study was designed to investigate the effects of inhibition of ODC during discrete prenatal periods on renal growth and function. We administered 5 doses of 500 mg/kg DFMO or saline s.c. to timed pregnant Sprague-Dawley rats at 12 hr intervals beginning on gestation days (GD) 11, 14, or 17. Half the dams were killed on GD 20 for fetal morphological analyses and half were allowed to go to term. Renal function was assessed on postnatal days (PD) 3, 6, 10, and 14 by tests of basal renal clearance and urinary concentrating ability, and on PD 42-44 we measured serum chemistries. All three gestational treatment regimens resulted in postnatal deficits in general growth. Only in the GD 11-13 treatment group was there evidence of embryotoxicity and neonatal renal pathophysiology. Fetal weights and urogenital morphology were altered following GD 14-16 treatment and there were persistent deficits of renal growth. GD 17-19 treatment was associated only with transient postnatal deficits of renal growth. Thus, inhibition of ODC during critical prenatal periods induced distinct developmental effects. However, there were no associations between impaired renal growth and function. These data indicate that general tissue growth is not always a predictor of physiological development and support the necessity of multifaceted approaches to the understanding of adverse developmental effects.     

Adipocytes and preadipocytes promote the proliferation of colon cancer cells in vitro

Obesity, a risk factor for colon cancer, is associated with elevated serum levels of leptin, a protein produced by adipocytes. The aim of the present study was to clarify the effects of adipose tissue on colon cancer proliferation by using cultured cell lines. To achieve this, colon cancer cells (CACO-2, T84, and HT29) were cocultured with adipose tissue, isolated mature adipocytes, and isolated preadipocytes in a three-dimensional collagen gel culture system. The adipocytes and preadipocytes used were isolated from C57BL/6J and leptin-deficient ob/ob mice. Proliferation of the cancer cells was evaluated by nuclear bromodeoxyuridine uptake. The adipose tissue, mature adipocytes, and preadipocytes isolated from C57BL/6J mice significantly increased the proliferation of the colon cancer cells. This trophic effect of mature adipocytes on the cancer cell lines was observed only for cells from lean littermates and not for those from ob/ob mice. In contrast, the trophic effect of preadipocytes was not abolished in ob/ob mice, and this finding was supported by the result that leptin had a trophic effect on cancer cells. In conclusion, adipocytes were able to enhance the proliferation of colon cancer cells in vitro, partly via leptin, suggesting that adipose tissues, including mature adipocytes and preadipocytes, may promote the growth of colorectal cancer.     

Effects of growth factors FGF2 and IGF2 on the development of parthenogenetic mouse embryos in utero and in vitro

We studied the effects of fibroblast growth factor 2 (FGF2) and insulin-like growth factor 2 (IGF2) on the development of parthenogenetic mouse embryos (CBA x C57BK/6)F1. The parthenogenetic embryos were treated in vitro during the preimplantation period and, at the blastocyst stage, transplanted into the uterus of pseudopregnant females. The addition of FGF2 at an optimal dose (2.5 ng/ml) to the culture medium increased twofold the number of embryos developed in utero to the somite stages as compared to the control: 18 and 43%, respectively. The parthenogenetic embryos (18-21 somites), treated and nontreated with FGF2 during the preimplantation period, were explanted for further development in vitro and treated with IGF2 at 2.5 micrograms/ml. As a result, many more parthenogenetic embryos (> 87%) of both groups developed in vitro to the stage of 30 or more somites as compared to the control (59%). The treatment of the parthenogenetic embryos with FGF2 alone at the preimplantation stages did not improve their development in vitro at the postimplantation stages. The results we obtained suggest that the treatment of parthenogenetic embryos in vitro with FGF2 during the preimplantation period increased twofold the number of somite embryos in utero, while their subsequent treatment in vitro with IGF2 leads to a significant prolongation of their development, as compared to the control.     

Physiological consequences of early neonatal growth retardation: effects of alpha-difluoromethylornithine on renal growth and function in the rat

The physiological consequences of early neonatal growth retardation in the kidney were investigated using alpha-difluoromethylornithine (DFMO), a specific irreversible inhibitor of ornithine decarboxylase (ODC), a key enzyme in the biosynthesis of polyamines. We administered by s.c. 500 mg/kg/day DFMO, or saline, to Sprague-Dawley rat pups from the day of birth through postnatal day (PD) 6 and evaluated renal function on PD 4, 7, 10, and 13 using tests of basal renal clearance and urinary concentrating ability. Kidney weights and gross pathology were also obtained. On PD 39, serum chemistries and organ weights were determined. In a second experiment, we evaluated concentrating ability on PD 7-10, and basal renal function, concentrating ability, diuretic response, serum chemistries, and organ weights on PD 132-140. DFMO selectively inhibited renal growth but did not inhibit glomerular and tubular functional maturation. In fact, the rates of filtration and reabsorption (per g renal tissue), and concentrating ability were increased in treated pups. These changes were associated with long-term effects on renal function, including uremia, glucosuria, and male-specific concentrating deficits in adulthood. Several hypotheses can be developed concerning the physiological mechanisms underlying these changes (e.g., altered renal urea metabolism), which in turn may reflect either a direct role of ODC in the regulation of maturation or secondary consequences of inhibition of ODC.(ABSTRACT TRUNCATED AT 250 WORDS)     

几丁聚糖对双歧杆菌体外生长的影响

目的观察几丁聚糖对双歧杆菌是否有增菌作用。方法应用分光光度比色法分析几丁聚糖对双歧杆菌体外生长作用的影响,并与中药黄芪对该菌的作用进行比较。结果与对照组比较,几丁聚糖和黄芪浓度分别为2.5 mg/mL和62.5 mg/mL时,对双歧杆菌有明显的增殖作用（P〈0.05）。比较两种药物在该浓度时对双歧杆菌的生长作用,虽然黄芪的增殖作用高于几丁聚糖,但差异无统计学意义（P〉0.05）。结论 2.5 mg/mL剂量的几丁聚糖体外能够促进双歧杆菌的生长。     

Effects of culture milieus on the development of mouse blastocysts in vitro

Summary Various culture milieus were examined for their support of mouse blastocyst development. Two important variables were the time at which human cord serum was added to the medium and the concentration of amino acids. In the best medium, Eagle's Minimum Essential Medium (fortified with six times the usual amino-acid concentration plus 20% fetal bovine serum, replaced after 48 hr with human cord serum), 83% of the blastocysts shed the zona pellucida, 58% developed to the early egg cylinder stage, 42% to the advanced egg cylinder stage and 22% attained the primitive streak stage after 6 to 8 days of culture. A preliminary account was given at the Tissue Culture Association Meeting in 1976 and the abstract published in its proceedings (1). This work was supported by MRC Grant No. MA4235.