Tylophorine reduces protein biosynthesis and rapidly decreases cyclin D1, inhibiting vascular smooth muscle cell proliferation in vitro and in organ culture.

BACKGROUND: Tylophorine (TYL) is an alkaloid with antiproliferative action in cancer cells. Vascular smooth muscle cell (VSMC) proliferation and neointima formation contribute to restenosis after percutaneous coronary interventions. HYPOTHESIS/PURPOSE: Our goal was to examine the potential of TYL to inhibit VSMC proliferation and migration, and to dissect underlying signaling pathways. STUDY DESIGN AND METHODS: TYL was administered to platelet-derived growth factor (PDGF-BB)-stimulated, serum-stimulated, quiescent and unsynchronized VSMC of rat and human origin. BrdU incorporation and resazurin conversion were used to assess cell proliferation. Cell cycle progression was analyzed by flow cytometry of propidium iodide-stained nuclei. Expression profiles of proteins and mRNAs were determined using western blot analysis and RT-qPCR. The Click-iT OPP Alexa Fluor 488 assay was used to monitor protein biosynthesis. RESULTS: TYL inhibited PDGF-BB-induced proliferation of rat aortic VSMCs by arresting cells in G1 phase of the cell cycle with an IC50 of 0.13 µmol/l. The lack of retinoblastoma protein phosphorylation and cyclin D1 downregulation corroborated a G1 arrest. Inhibition of proliferation and cyclin D1 downregulation were species- and stimulus-independent. TYL also decreased levels of p21 and p27 proteins, although at later time points than observed for cyclin D1. Co-treatment of VSMC with TYL and MG132 or cycloheximide (CHX) excluded proteasome activation by TYL as the mechanism of action. Comparable time-dependent downregulation of cyclin D1, p21 and p27 in TYL- or CHX-treated cells, together with decreased protein synthesis observed in the Click-iT assay, suggests that TYL is a protein synthesis inhibitor. Besides proliferation, TYL also suppressed migration of PDGF-activated VSMC. In a human saphenous vein organ culture model for graft disease, TYL potently inhibited intimal hyperplasia. CONCLUSION: This unique activity profile renders TYL an interesting lead for the treatment of vasculo-proliferative disorders, such as restenosis.

Marsdenia Tenacissima Extract Inhibits Proliferation and Promotes Apoptosis in Human Ovarian Cancer Cells.

BACKGROUND Marsdenia tenacissima extract (MTE) is a traditional Chinese medicine that can be effectively used against various cancers. However, to the best of our knowledge, its role in ovarian cancer is not known. This study investigated the effects of MTE on human ovarian cancer SKOV3 cells. MATERIAL AND METHODS The viability and cell cycle of SKOV3 cells were assessed using the cell counting kit-8 (CCK-8) and propidium Iodide (PI) staining kit, respectively. Cell apoptosis and mitochondrial membrane potential (MMP) were detected by flow cytometry. The expression levels of proliferation-related and apoptosis-related factors were tested by quantitative real-time PCR (qRT-PCR) and Western blot assays, respectively. RESULTS We found that MTE markedly reduced the viability of SKOV3 cells in dose-dependent and time-dependent manners. MTE induced cell cycle arrest by downregulating the levels of cyclin D1and cyclin B1. MTE (10, 20, and 40 mg/mL) markedly increased apoptosis rates (2.77±0.6%, 4.95±0.97%, and 12.16±0.69%, respectively), and enhanced the loss of MMP. MTE obviously downregulated the expression of B cell lymphoma-2 (Bcl-2) and upregulated the expression levels of fibroblast-associated (Fas), Fas ligand (FasL), cleaved cysteinyl aspartate-specific proteinas-3 (caspase-3), and Bcl-2-associated X protein (Bax) compared to the control group. In addition, the expressions of phosphorylated mammalian target of rapamycin (p-mTOR), phosphorylated protein kinase B (p-AKT), and phosphorylated-phosphatidylinositol 3 kinase (p-PI3K) were decreased by MTE. CONCLUSIONS MTE inhibited proliferation and induced apoptosis of SKOV3 cells. The depression of the PI3K/AKT/mTOR pathway may augment the protective effect of MTE. Thus, MTE might be expected to be a new drug for curing ovarian cancer.

Shikonin enhances sensitization of gefitinib against wild-type EGFR non-small cell lung cancer via inhibition PKM2/stat3/cyclinD1 signal pathway.

AIMS: Mutant EGFR Non-small cell lung cancer has benefit from gefitinib, but it has limited effect for wild-type EGFR tumors. Shikonin, a natural naphthoquinone isolated from a traditional Chinese medicine, the plant Lithospermum erythrorhizon (zicao), not only can inhibit the tumor growth, but also overcome cancer drug resistance. Our aim is to investigate whether shikonin can enhance antitumor effect of gefitinib in EGFR wild-type lung cancer cells in vitro and in vivo. MATERIALS AND METHODS: CCK-8 was used to determine the proliferation of EGFR wild-type non-small cell lung cancer. Apoptosis and cell cycle were detected by flow cytometry. PKM2, STAT3, p-STAT3 and cyclinD1 were detected by Western blot. A549 tumor model was established to observe the antitumor effect of shikonin combination with gefitinib in vivo. KEY FINDINGS: The results showed that combination of shikonin with gefitinib exhibited synergistic antitumor effect in vitro and in vivo. Its potential molecular mechanisms may be associated with inhibition of PKM2/STAT3/cyclinD1. SIGNIFICANCE: These results provide a promising therapeutic approach for the treatment of wild-type EGFR non-small cell lung cancer.

[Effect of RAD18-siRNA on proliferation and chemotherapy sensitivity of human esophageal squamous cell carcinoma ECA-109 cells].

Objective: To investigate the effect of RAD18-siRNA on cell proliferation and chemotherapy sensitivity of esophageal squamous cell carcinoma (ESCC) ECA-109 cells. Methods: RAD18-siRNA was transfected into human ECA-109 cells by Lipofectamine 3000. Quantitative PCR and Western blot were performed to detect RAD18 and CyclinD1 expression; CCK-8 assay was used to determine cell proliferation and chemotherapy drug sensitivity; flow cytometry was used to determine cell cycle. Correlation between RAD18 and CyclinD1 mRNA expression was analyzed by Pearson's correlation. Results: Compared with non-transfected cells, the expression of RAD18 in RAD18-siRNA group was significantly decreased (P<0.05). The cell proliferation was inhibited (P<0.05) and the cell number of G1 phase was increased, G2/M phase cells decreased (P<0.05) in RAD18-siRNA group. After treatment with different concentrations of cisplatin or 5-FU, the survival rate of the two cell groups was reduced (all P<0.05), and the IC50 of RAD18-siRNA group was significantly lower than that of non-transfected group (P<0.05). The mRNA expression of RAD18 was positively correlated with CyclinD1 expression in ESCC tissues(r=0.478, P<0.01). Conclusion: Down-regulated expression of RAD18 can decrease the cell proliferation and increase chemo-sensitivity of ESCC cells, and CyclinD1 may participate in the process.

[Anti-tumor effects of 13-cis-retinoic acid combined with interferon α-2b in animal model of mantle cell lymphoma].

Objective: To determine the anti-tumor effects of 13-cis-retinoic acid (13cRA) combined with interferonα-2b (IFNα-2b) in mantle cell lymphoma (MCL) animal model. Methods: The animal model of MCL was established by introducing Jeko-1 cell line into severe combined immunodeficiency disease mice. The successfully tumor-developed mice were assigned to different groups treated with negative control group (solvents), 13cRA (high dose: 200mg/kg; middle dose: 100mg/kg; low dose: 50 mg/kg) alone, IFNα-2b alone or combination of different dose of 13cRA with IFNα-2b, and positive control group (bortezomib, rituximab, cyclophosphamide), respectively. Variations of tumor volume were observed regularly. The relative tumor proliferation rate and tumor inhibition rate were calculated. Immunohistochemistry stain was used to detect the Ki-67 expression and TUNEL was applied to measure the apoptosis of tumor cells. Furthermore, the levels of Cyclin D1, caspase 9 and Rb protein were measured by Western-blot method. Results: ① The relative tumor proliferation rates (T/C%)were 30%, 37%, 32% and 33% in middle dose, high dose groups of 13cRA as well as their combination with IFN α-2b, respectively. ② Comparing with the negative control, both 13cRA at different doses and its combination with IFNα-2b remarkably inhibited the tumor growth (P<0.05), while no statistic significance existed in different dose group of 13cRA. IFN-α 2b alone didn't demonstrate the tumor-inhibition effects (P>0.05). Middle dose of 13cRA and its combination with IFN-α-2b demonstrated relatively high tumor-inhibition effects (59.2% and 62.6% respectively), which were similar to the effects in positive control (69.4%). ③ There was no statistic difference of Ki-67 in each experimental group. ④ Comparing with negative control group, all doses of 13cRA and their combinations with IFNα-2b remarkably increased the apoptosis (P<0.05), similar to the positive control group (P>0.05). However, IFNα-2b alone didn' t promote the apoptosis of tumor tissue (P=0.098). ⑤ Comparing with negative control group, IFNα-2b combined with each dose of 13cRA significantly decreased the levels of cycling D1 and procaspase-9, while increased the level of cleaved caspase-9 (P<0.05), which were similar to the positive control group (P>0.05). Nevertheless, 13cRA alone didn't demonstrate such effects. Conclusion: In the MCL animal model, IFNα-2b alone showed no effects, but combined with IFNα-2b, 13cRA displayed anti-tumor effects at different doses. The anti-tumor mechanism of 13cRA combined with IFNα-2b was probably down-regulation of the cyclin D1 expression, inhibition of cell proliferation and induction of apoptosis by activating caspase-9.

Photoperiodic and Diurnal Regulation of WNT Signaling in the Arcuate Nucleus of the Female Djungarian Hamster, Phodopus sungorus.

The WNT pathway was shown to play an important role in the adult central nervous system. We previously identified the WNT pathway as a novel integration site of the adipokine leptin in mediating its neuroendocrine control of metabolism in obese mice. Here we investigated the implication of WNT signaling in seasonal body weight regulation exhibited by the Djungarian hamster (Phodopus sungorus), a seasonal mammal that exhibits profound annual changes in leptin sensitivity. We furthermore investigated whether crucial components of the WNT pathway are regulated in a diurnal manner. Gene expression of key components of the WNT pathway in the hypothalamus of hamsters acclimated to either long day (LD) or short day (SD) photoperiod was analyzed by in situ hybridization. We detected elevated expression of the genes WNT-4, Axin-2, Cyclin-D1, and SFRP-2, in the hypothalamic arcuate nucleus, a key energy balance integration site, during LD compared with SD as well as a diurnal regulation of Axin-2, Cyclin-D1, and DKK-3. Investigating the effect of photoperiod as well as leptin on the activation (phosphorylation) of the WNT coreceptor LRP-6-(Ser1490) by immunohistochemistry, we found elevated activity in the arcuate nucleus during LD relative to SD as well as after leptin treatment (2 mg/kg body weight). These findings indicate that differential WNT signaling may be associated with seasonal body weight regulation and is partially regulated in a diurnal manner in the adult brain. Furthermore, they suggest that this pathway plays a key role in the neuroendocrine regulation of body weight and integration of the leptin signal.

Alpha-ketoglutarate (AKG) inhibits proliferation of colon adenocarcinoma cells in normoxic conditions.

BACKGROUND AND OBJECTIVE: Alpha-ketoglutarate (AKG), a key intermediate in Krebs cycle, is an important biological compound involved in the formation of amino acids, nitrogen transport, and oxidation reactions. AKG is already commercially available as a dietary supplement and its supplementation with glutamine, arginine, or ornithine alpha-ketoglutarate has been recently considered to improve anticancer immune functions. It is well documented that AKG treatment of Hep3B hepatoma cells in hypoxia induced HIF-alpha (hypoxia-inducible factor) degradation and reduced vascular endothelial growth factor (VEGF) synthesis. Moreover, AKG showed potent antitumor effects in murine tumor xenograft model, inhibiting tumor growth, angiogenesis, and VEGF gene expression. However, the mechanisms of its anticancer activity in normoxia have not been examined so far. RESULTS: Here, we report that in normoxia, AKG inhibited proliferation of colon adenocarcinoma cell lines: Caco-2, HT-29, and LS-180, representing different stages of colon carcinogenesis. Furthermore, AKG influenced the cell cycle, enhancing the expression of the inhibitors of cyclin-dependent kinases p21 Waf1/Cip1 and p27 Kip1. Moreover, expression of cyclin D1, required in G1/S transmission, was decreased, which accompanied with the significant increase in cell number in G1 phase. AKG affected also one the key cell cycle regulator, Rb, and reduced its activation status. CONCLUSION: In this study for the first time, the antiproliferative activity of AKG on colon adenocarcinoma Caco-2, HT-29, and LS-180 cells in normoxic conditions was revealed. Taking into consideration an anticancer activity both in hypoxic and normoxic conditions, AKG may be considered as a new potent chemopreventive agent.

Curcumin, but not Prima-1, decreased tumor cell proliferation in the syngeneic murine orthotopic bladder tumor model.

OBJECTIVE: Cigarette smoking is the main risk factor for bladder cancer development. Among the mediators of this effect of smoking is nuclear factor-kappa B. Curcumin suppresses cellular transformation by downregulating the activity of nuclear factor-kappa B. Prima-1 is a compound that induces apoptosis in human tumor cells, restoring the function of mutant p53. Our study aimed to evaluate the effects of curcumin and prima-1 in an animal model of bladder cancer. METHODS: Tumor implantation was achieved in six- to eight-week-old female C57BL/6 mice by introducing MB49 bladder cancer cells into the bladder. Intravesical treatment with curcumin and Prima-1 was performed on days 2, 6, 10, and 14. On day 15, the animals were sacrificed. Immunohistochemistry was used to determine the expression of cyclin D1, Cox-2, and p21. Cell proliferation was examined using PCNA. RESULTS: Animals treated with curcumin exhibited a higher degree of necrosis than animals in other groups. Immunohistochemistry showed reduced expression of cyclin D1 in the curcumin-treated group. All of the cells in mice treated with curcumin were p21 positive, suggesting that the p53 pathway is induced by this compound. Prima-1 did not induce any change in tumor size, necrosis, cell proliferation, or the expression of proteins related to the p53 pathway in this animal model. CONCLUSION: Curcumin showed activity in this animal bladder cancer model and probably acted via the regulation of nuclear factor-kappa B and p53. Therefore, curcumin is a good choice for the use in clinical trials to treat superficial bladder cancer as an alternative to bacillus Calmette-Guerin. In contrast, Prima-1 does not seem to have an effect on bladder cancer.

Curcumin, but not Prima-1, decreased tumor cell proliferation in the syngeneic murine orthotopic bladder tumor model

OBJECTIVE: Cigarette smoking is the main risk factor for bladder cancer development. Among the mediators of this effect of smoking is nuclear factor-kappa B. Curcumin suppresses cellular transformation by downregulating the activity of nuclear factor-kappa B. Prima-1 is a compound that induces apoptosis in human tumor cells, restoring the function of mutant p53. Our study aimed to evaluate the effects of curcumin and prima-1 in an animal model of bladder cancer. METHODS: Tumor implantation was achieved in six- to eight-week-old female C57BL/6 mice by introducing MB49 bladder cancer cells into the bladder. Intravesical treatment with curcumin and Prima-1 was performed on days 2, 6, 10, and 14. On day 15, the animals were sacrificed. Immunohistochemistry was used to determine the expression of cyclin D1, Cox-2, and p21. Cell proliferation was examined using PCNA. RESULTS: Animals treated with curcumin exhibited a higher degree of necrosis than animals in other groups. Immunohistochemistry showed reduced expression of cyclin D1 in the curcumin-treated group. All of the cells in mice treated with curcumin were p21 positive, suggesting that the p53 pathway is induced by this compound. Prima-1 did not induce any change in tumor size, necrosis, cell proliferation, or the expression of proteins related to the p53 pathway in this animal model. CONCLUSION: Curcumin showed activity in this animal bladder cancer model and probably acted via the regulation of nuclear factor-kappa B and p53. Therefore, curcumin is a good choice for the use in clinical trials to treat superficial bladder cancer as an alternative to bacillus Calmette-Guerin. In contrast, Prima-1 does not seem to have an effect on bladder cancer.

Anti-leukaemic effects of two extract types of Lactuca sativa correlate with the activation of Chk2, induction of p21, downregulation of cyclin D1 and acetylation of alpha-tubulin.

The water extract of the lettuce Lactuca sativa, but not the ethyl acetate extract, inhibited the growth of HL-60 leukaemia cells and MCF-7 breast cancer cells. This correlated with the activation of checkpoint kinase 2 (Chk2), the induction of the tumour suppressor p21, and the severe downregulation of the proto-oncogene cyclin D1. The ethyl acetate extract, but not the water extract, induced HL-60 cell death, which correlated with the acetylation of alpha-tubulin. The acetylation of alpha-tubulin is indicative for microtubuli stabilisation such as induced by taxol. The calculated amount for human intake would require approximately 3 kg lettuce to reach the required concentration shown to inhibit 50% HL-60 proliferation.

Polysaccharide peptide mediates apoptosis by up-regulating p21 gene and down-regulating cyclin D1 gene.

The use of herbal medicine is a common practice among Chinese women with breast cancer. Yunzhi (Voriolus versicolor), a substance that is regarded as a biological response modifier, is frequently used. The aim of the present study is to evaluate the anti-proliferative action of, Yunzhi, polysaccharide peptide (PSP), on breast cancer cells. Breast cancer cells (MDA-MB-231) were cultured with and without PSP for 7 days. Cell growth at 24, 72, 120 and 168 hours was measured by Cell Proliferation Reagent (WST-1). Cells treated with PSP were found to have a significant reduction in cell proliferation as compared to controls after 72 hours of incubation. This lasted for 168 hours. When the effect of PSP on apoptosis was studied by the TdT-mediated X-dUTP nick end-labeling (TUNEL) assay, we found that PSP had a significant effect upon apoptosis from 24 hours onward. Immunostaining showed that PSP increased p21 expression and decreased cyclin D1 expression. In conclusion, PSP is effective in inhibiting cell proliferation through apoptosis. The mechanism for the apoptosis may be through up-regulation of p21 and down-regulation of cyclin D1.

Ursodeoxycholic acid and F(6)-D(3) inhibit aberrant crypt proliferation in the rat azoxymethane model of colon cancer: roles of cyclin D1 and E-cadherin.

We have previously demonstrated that ursodeoxycholic acid(UDCA) and a fluorinated analogue of vitamin D(3), F(6)-D(3),inhibited colonic carcinogenesis in the azoxymethane (AOM) model. Generalized colonic mucosal hyperproliferation and aberrant crypt foci (ACF) are intermediate biomarkers of colon cancer. Using these biomarkers, in this study we examined the anticarcinogenic mechanisms of these chemopreventive agents. Rats were maintained on AIN-76A chow or supplemented with 0.4% UDCA or F(6)-D(3) (2.5 nmol/kg chow) and treated weekly with AOM 20 mg i.p./kg wt or saline x 2 weeks. F(6)-D(3) was continued for an additional 2 weeks and UDCA for the duration of the study. At 40 weeks, animals received bromodeoxyuridine (BrdUrd) i.p. 2 h before sacrifice. A portion of each tumor was fixed in formalin and the remainder flash frozen. Colons were divided longitudinally and half-fixed in formalin and half in ethanol. The size and location of methylene blue-stained ACF were recorded. Cell proliferation (BrdUrd labeling) and apoptosis (terminal deoxynucleotidyl transferase-mediated nick end labeling assay) were measured in colonic crypts and tumors. Protein expression levels of several regulators of cell proliferation were analyzed by immunostaining and Western blotting. Colonic crypt cyclin D1 and E-cadherin mRNA levels were measured by real-time PCR. In saline injected controls, neither UDCA nor F(6)-D(3) alone had any effect on cytokinetic parameters or on the expression of mitogenic regulators. AOM significantly increased the proliferation (percentage of BrdUrd-positive cells) of both ACF (23.1 +/- 1.7%) and non-ACF crypts (17.6 +/- 1.6%), compared with normal colonic crypts (4.5 +/- 0.8%; P < 0.05). This hyperproliferation was accompanied by a 5-fold increase in cyclin D1 and >50% decrease in E-cadherin protein (P < 0.05) in ACF, both of which are predicted to be growth-enhancing alterations. UDCA and F(6)-D(3) significantly (P < 0.05) inhibited AOM-induced crypt cell hyperproliferation, ACF development, and tumor burden. These chemopreventive agents also significantly blocked AOM-induced alterations in cyclin D1 and E-cadherin protein in ACF and tumors. In ACF, changes in mRNA levels of cyclin D1, but not E-cadherin, paralleled alterations in protein expression. Cyclooxygenase-2 and inducible nitric oxide synthase were increased in AOM tumors but not in ACF, and these changes were blocked by UDCA and F(6)-D(3). UDCA and F(6)-D(3) significantly inhibited ACF development and hyperproliferation, in part, by preventing carcinogen-induced alterations in cyclin D1 and E-cadherin. In established tumors, UDCA and F(6)-D(3) also limited inductions of cyclooxygenase-2 and inducible nitric oxide synthase, which together with their effects on cyclin D1 and E-cadherin, contribute to their chemopreventive actions.

[Effects of tea polyphenols and tea pigments on cell cycle regulators in rat liver precancerous lesions].

OBJECTIVES: This study is to investigate the effects of tea polyphenols and tea pigments on cell cycle regulators in rat liver precancerous lesions. METHODS: The modified Solt-Farber precancerous liver rat model was used. Rats were given water, tea polypheol solution (0.1%) or tea pigment solution (0.1%) throughout the whole experiment (56 days). Cyclin D1, P21(WAF1/CIP1), GADD45 and PCNA protein expression were detected by Western blotting and the RT-PCR method was applied to study the expression of Cdk4. RESULTS: Cyclin D1, Cdk4 and PCNA expressions were significantly inhibited, and the expression of P21(WAF1/CIP1) and GADD45 were significantly induced by tea polyphenols and tea pigments treatments. CONCLUSION: Tea polyphenols and tea pigments induced cell cycle arrest and inhibited cell proliferation by regulating cell cycle regulators.

Extract from the leaves of Toona sinensis roemor exerts potent antiproliferative effect on human lung cancer cells.

Recent study indicated that the components of Toona sinensis Roemor have potent anti-inflammatory and analgesic effects. These components have also been reported to inhibit the growth of boils in vivo. In this study, we investigated the effect of crude extract from the leaves of Toona sinensis Roemor on the proliferation of A549 lung cancer cells. We found that the extract effectively blocked cell cycle progression by inhibiting the expression of cyclin D1 and E in A549 cells. Additionally, incubation of the extract led to activation of caspase-3-like proteases and apoptotic cell death. Conversely, the extract did not show any significant cytotoxic effect on primarily cultured human foreskin fibroblasts or MRC-5 human lung fibroblasts. Therefore, antiproliferative action of the extract is specific for tumor cells. Our results suggest that the components of Toona sinensis Roemor have potent anticancer effects in vitro and identification of the useful components in the extract may lead to the development of a novel class of anticancer drugs.

Soluble collagen VI drives serum-starved fibroblasts through S phase and prevents apoptosis via down-regulation of Bax.

We previously showed that soluble, pepsin-solubilized collagen VI increases de novo DNA synthesis in serum-starved HT1080 and 3T3 fibroblasts up to 100-fold compared with soluble collagen I, reaching 80% of the stimulation caused by 10% fetal calf serum. Here we show that collagen VI also inhibits apoptotic cell death in serum-starved cells as evidenced by morphological criteria, DNA laddering, complementary apoptosis assays (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling, enzyme-linked immunosorbent assay, and fluorescence-activated cell sorting), and quantification of apoptosis-regulating proteins. In the presence of starving medium alone or collagen I, the proapoptotic Bax was up-regulated 2-2.5-fold, compared with soluble collagen VI and fetal calf serum, whereas levels of the antiapoptotic Bcl-2 protein remained unaffected. In accordance with its potent stimulation of DNA synthesis, soluble collagen VI carries serum-starved HT1080 and Balb 3T3 fibroblasts through G(2) as shown by fluorescence-activated cell sorting analysis, whereas cells exposed to medium and collagen I where arrested at G(1)-S. This was accompanied by a 2-3-fold increase in cyclin A, B, and D1 protein expression. Collagen VI-induced inhibition of apoptotic cell death may be operative during embryogenesis, wound healing, and fibrosis when elevated tissue and blood levels of collagen VI are observed, thus initiating a feedback loop of mesenchymal cell activation and proliferation.