Amygdalin Influences Bladder Cancer Cell Adhesion and Invasion In Vitro

The cyanogenic diglucoside amygdalin, derived from Rosaceae kernels, is employed by many patients as an alternative anti-cancer treatment. However, whether amygdalin indeed acts as an anti-tumor agent is not clear. Metastasis blocking properties of amygdalin on bladder cancer cell lines was, therefore, investigated. Amygdalin (10 mg/ml) was applied to UMUC-3, TCCSUP or RT112 bladder cancer cells for 24 h or for 2 weeks. Tumor cell adhesion to vascular endothelium or to immobilized collagen as well as tumor cell migration was examined. Effects of drug treatment on integrin α and β subtypes, on integrin-linked kinase (ILK) and total and activated focal adhesion kinase (FAK) were also determined. Integrin knock-down was carried out to evaluate integrin influence on migration and adhesion. A 24 h or 2 week amygdalin application distinctly reduced tumor cell adhesion and migration of UMUC-3 and RT112 cells. TCCSUP adhesion was also reduced, but migration was elevated under amygdalin. Integrin subtype expression was significantly and specifically altered by amygdalin depending on the cell line. ILK was moderately, and activated FAK strongly, lost in all tumor cell lines in the presence of amygdalin. Knock down of β1 integrin caused a significant decrease in both adhesion and migration of UMUC-3 cells, but a significant increase in TCCSUP adhesion. Knock down of β4 integrin caused a significant decrease in migration of RT112 cells. Since the different actions of amygdalin on the different cell lines was mirrored by β1 or β4 knock down, it is postulated that amygdalin influences adhesion and migratory properties of bladder cancer cells by modulating β1 or β4 integrin expression. The amygdalin induced increase in TCCSUP migratory behavior indicates that any anti-tumor benefits from amygdalin (seen with the other two cell lines) may depend upon the cancer cell type.     

Expression of cyclins and cdks throughout murine carcinogenesis.

The overexpression and/or amplification of cell cycle regulating genes is an important factor in the progression of cancer. Recent attention has been focused on several cyclin and cdks genes whose expression were increased in many types of tumor. In this study, we investigated the expression kinetics of cyclins A, B, D1, E and cdks 1, 2, 4, 6 by RT-PCR coupled with densitometry and correlated to the growth fraction (percentage of S cells). This analysis was performed using an experimental murine leukemic model, generated by in vivo administration of murine clonogenic cells Wehi-3b injected into balb-c mice. Differential expression of cyclins and cdks was observed between normal and tumoral cells with different patterns of expression between G1 and G2M cyclins-cdks. G1 cyclins cdks expression was significantly increased in tumor cells when compared to normal cells. In the same manner, G2M cyclins cdks expression was only observed in tumor cells at a lower level than for G1 cyclins cdks, but not detected in normal cells. These differences correlated with the growth fraction for both the G1 cyclins cdks (r = 0.91, 0.94, 0.85, 0.90 and 0.96 for cyclin D1, cyclin E, cdk2, cdk4 and cdk6, respectively) and the G2M cyclins cdks (r = 0.96, 0.97 and 0.93 for cyclins A, B and cdkl respectively). Analysis of cyclins cdks expression kinetics during tumoral progression shows that cyclins A, B and cdkl were expressed from the 12th day on of disease, increased until the death of the animals and correlated with the growth fraction (r = 0.94, 0.95 and 0.97 for cyclins A, B and cdk1 respectively) (n = 20). Overexpression of other cyclins cdks were observed, from the 6th day on for cyclin D1, the 12th day for cdk2 and cdk4, the 15th day for cdk6 and the 20th day for cyclin E. These increases persisted during tumoral progression and correlated with the growth fraction (r = 0.85, 0.94, 0.93, 0.96, and 0.98 for cyclin D1, cyclin E, cdk2, cdk4 and cdk6, respectively) (n = 20). Our results demonstrated that G1 and G2-M cyclins cdks mRNA levels were increased at approximately the same time of maximal tumor growth. Only cyclin D1 overexpression occured at the initiation of tumoral development, and could therefore be considered as an early marker of cell proliferation.     

Cyclin D3 is dispensable for human diffuse large B-cell lymphoma survival and growth: evidence for redundancy with cyclin E

Genomic changes disrupting the expression of cyclin D3 are associated with aberrant growth of several human B-lymphoid malignancies. We demonstrate that the human diffuse large B-cell lymphoma (DLBCL), OCI-LY18 (LY18) expresses cyclin D3 but not cyclins D1 and D2. RNA interference was used to deplete cyclin D3 from LY18 cells. Surprisingly, knockdown of cyclin D3 did not inhibit pRb phosphorylation on cdk4/6- and cdk2-specific residues or measurably affect viability and proliferation. These results suggest that cyclin D3 is dispensable in LY18 cell proliferation and survival. Similar results were obtained following depletion of cyclin E. By contrast, combined knockdown of cyclins D3 and E had substantial consequences leading to G1-phase arrest and inhibition of proliferation. Whereas cell cycle distribution was not affected following individual depletion of cdk4, cdk6, or cdk2, the combined knockdown of cdk4 and cdk6 led to accumulation of LY18 cells in G1-phase of the cell cycle and inhibition of proliferation. Likewise treatment of LY18 cells with 2-Bromo-12,13-dihydro-5H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7(6H)-dione, a selective inhibitor of cdk4/6, led to inhibition of proliferation. Taken together, these results uncover a built-in redundancy with cyclins D3 and E for G1-S progression. Moreover these findings highlight the rationale for simultaneous disruption of cdk4/6 as a potential therapeutic cancer strategy.     

Cyclin D1 is dispensable for G1 control in retinoblastoma gene-deficient cells independently of cdk4 activity.

To elucidate the regulator-versus-target relationship in the cyclin D1/cdk4/retinoblastoma protein (pRB) pathway, we examined fibroblasts from RB-1 gene-deficient and RB-1 wild-type littermate mouse embryos (ME) and in human tumor cell lines that differed in the status of the RB-1 gene. The RB+/+ and RB-/- ME fibroblasts expressed similar protein levels of D-type cyclins, cdk4, and cdk6, showed analogous spectra and abundance of cellular proteins complexed with cdk4 and/or cyclins D1 and D2, and exhibited comparable associated kinase activities. Of the two human cell lines established from the same sarcoma biopsy, the RB-positive SKUT1B cells contained cdk4 that was mainly associated with D-type cyclins, contrary to a predominant cdk4-p16INK4 complex in the RB-deficient SKUT1A cells. Antibody-mediated neutralization of cyclin D1 arrested the RB-positive ME and SKUT1B cells in G1, whereas this cyclin appeared dispensable in the RB-deficient ME and SKUT1A cells. Lack of requirement for cyclin D1 therefore correlated with absence of functional pRB, regardless of whether active cyclin D1/cdk4 holoenzyme was present in the cells under study. Consistent with a potential role of cyclin D/cdk4 in phosphorylation of pRB, monoclonal anti-cyclin D1 antibodies supporting the associated kinase activity failed to significantly affect proliferation of RB-positive cells, whereas the antibody DCS-6, unable to coprecipitate cdk4, efficiently inhibited G1 progression and prevented pRB phosphorylation in vivo. These data provide evidence for an upstream control function of cyclin D1/cdk4, and a downstream role for pRB, in the order of events regulating transition through late G1 phase of the mammalian cell division cycle.     

Inhibition of G1 cyclin-dependent kinase activity during growth arrest of human breast carcinoma cells by prostaglandin A2.

Prostaglandin A2 (PGA2) potently inhibits cell proliferation and suppresses tumor growth in vivo, but little is known regarding the molecular mechanisms mediating these effects. Here we demonstrate that treatment of breast carcinoma MCF-7 cells with PGA2 leads to G1 arrest associated with a dramatic decrease in the levels of cyclin D1 and cyclin-dependent kinase 4 (cdk4) and accompanied by an increase in the expression of p21. We further show that these effects occur independent of cellular p53 status. The decline in cyclin D and cdk4 protein levels is correlated with loss in cdk4 kinase activity, cdk2 activity is also significantly inhibited in PGA2-treated cells, an effect closely associated with the upregulation of p21. Immunoprecipitation experiments verified that p21 was indeed complexed with cdk2 in PGA2-treated cells. Additional experiments with synchronized MCF-7 cultures stimulated with serum revealed that treatment with PGA2 prevents the progression of cells from G1 to S. Accordingly, the kinase activity associated with cdk4, cyclin E, and cdk2 immunocomplexes, which normally increases following serum addition, was unchanged in PGA2-treated cells. Furthermore, the retinoblastoma protein (Rb), a substrate of cdk4 and cdk2 whose phosphorylation is necessary for cell cycle progression, remains underphosphorylated in PGA2-treated serum-stimulated cells. These findings indicate that PGA2 exerts its growth-inhibitory effects through modulation of the expression and/or activity of several key G1 regulatory proteins. Our results highlight the chemotherapeutic potential of PGA2, particularly for suppressing growth of tumors lacking p53 function.     

Inhibition of G1 phase cyclin dependent kinases by transforming growth factor β1

Transforming growth factor β1 (TGFβ1) inhibits epithelial cell proliferation late in the G1 phase of the cell cycle. We examined the effect of TGFβ1 on known late G1 cell cycle regulators in an attempt to determine the molecular mechanism of growth inhibition by this physiological inhibitor. The results demonstrate the TGFβ1 inhibits the late G1 and S phase specific histone H1 kinase activity of p33^cdk2. This inhibitiion is not dur to TGFβ1's effect on p33^cdk2 synthesis, but rather due to its negative effect on the late G1 phosphorylation of p33^cdk2. It is also shown that TGFβ1 inhibits both late G1 cyclin A and cyclin E associated histon H1 kinase activities. The inhibitor has no effects on the synthesis of cyclin E but to inhibit the synthesis of cyclin A protein in a cell cycle dependent manner. If TGFβ1 is added to cells which have progressed futher than 8 hours into G1, then it is without inhibitory effect on cyclin A synthesis. These effect on TGFβ1 on late G1 cell cycle regulators correlate well with its inhibitory effects on cellular growth and suggest that these G1 cyclin dependent kinases might serve as targets for TGFβ1-mediated growth arrest.     

Estrogen-dependent cyclin E-cdk2 activation through p21 redistribution.

In order to elucidate the mechanisms by which estrogens and antiestrogens modulate the growth of breast cancer cells, we have characterized the changes induced by estradiol that occur during the G1 phase of the cell cycle of MCF-7 human mammary carcinoma cells. Addition of estradiol relieves the cell cycle block created by tamoxifen treatment, leading to marked activation of cyclin E-cdk2 complexes and phosphorylation of the retinoblastoma protein within 6 h. Cyclin D1 levels increase significantly while the levels of cyclin E, cdk2, and the p21 and p27 cdk inhibitors are relatively constant. However, the p21 cdk inhibitor shifts from its association with cyclin E-cdk2 to cyclin D1-cdk4, providing an explanation for the observed activation of the cyclin E-cdk2 complexes. These results support the notion that cyclin D1 has an important role in steroid-dependent cell proliferation and that estrogen, by regulating the activities of G1 cyclin-dependent kinases, can control the proliferation of breast cancer cells.     

New Castanospermine Glycoside Analogues Inhibit Breast Cancer Cell Proliferation and Induce Apoptosis without Affecting Normal Cells

sp²-Iminosugar-type castanospermine analogues have been shown to exhibit anti-tumor activity. However, their effects on cell proliferation and apoptosis and the molecular mechanism at play are not fully understood. Here, we investigated the effect of two representatives, namely the pseudo-S- and C-octyl glycoside 2-oxa-3-oxocastanospermine derivatives SO-OCS and CO-OCS, on MCF-7 and MDA-MB-231 breast cancer and MCF-10A mammary normal cell lines. We found that SO-OCS and CO-OCS inhibited breast cancer cell viability in a concentration- and time-dependent manner. This effect is specific to breast cancer cells as both molecules had no impact on normal MCF-10A cell proliferation. Both drugs induced a cell cycle arrest. CO-OCS arrested cell cycle at G1 and G2/M in MCF-7 and MDA-MB-231cells respectively. In MCF-7 cells, the G1 arrest is associated with a reduction of CDK4 (cyclin-dependent kinase 4), cyclin D1 and cyclin E expression, pRb phosphorylation, and an overexpression of p21^Waf1/Cip1. In MDA-MB-231 cells, CO-OCS reduced CDK1 but not cyclin B1 expression. SO-OCS accumulated cells in G2/M in both cell lines and this blockade was accompanied by a decrease of CDK1, but not cyclin B1 expression. Furthermore, both drugs induced apoptosis as demonstrated by the increased percentage of annexin V positive cells and Bax/Bcl-2 ratio. Interestingly, in normal MCF-10A cells the two drugs failed to modify cell proliferation, cell cycle progression, cyclins, or CDKs expression. These results demonstrate that the effect of CO-OCS and SO-OCS is triggered by both cell cycle arrest and apoptosis, suggesting that these castanospermine analogues may constitute potential anti-cancer agents against breast cancer.     

苦杏仁苷介导人肝癌细胞HuH-7凋亡的实验研究

摘要 目的：通过体内体外实验探讨苦杏仁苷在肝癌中的抗肿瘤作用。方法：MTT 法检测不同浓度的苦杏仁苷对肝癌 HuH-7细胞存活率的影响;DAPI染色法观察苦杏仁苷介导HuH-7细胞的凋亡形态学变化;流式细胞术检测苦杏仁苷干预后HuH-7细胞凋亡率变化;Western Blot法检测细胞凋亡相关蛋白Bax、Bcl-2的表达,计算Bax/Bcl-2的比值。建立裸鼠HuH-7细胞移植瘤模型,观察苦杏仁苷对荷瘤裸鼠移植瘤体积的影响。结果：体外实验结果证实苦杏仁苷能够诱导人肝癌HuH-7细胞凋亡的发生（P<0.05）。随着苦杏仁苷浓度的增加,HuH-7细胞的存活率降低,凋亡率升高,干预后Bax/Bcl-2比值明显升高（P<0.05）。体内实验结果表明,裸鼠HuH-7细胞移植瘤的体积增长速度减慢（P<0.05）。结论：苦杏仁苷能够诱导人肝癌HuH-7细胞和裸鼠HuH-7细胞移植瘤细胞发生凋亡,减缓肿瘤生长,从而发挥抗肿瘤作用。     

Cell cycle dysregulation by green tea polyphenol epigallocatechin-3-gallate

Epidemiological, in vitro cell culture, and in vivo animal studies have shown that green tea or its constituent polyphenols, particularly its major polyphenol epigallocatechin-3-gallate (EGCG) may protect against many cancer types. In earlier studies, we showed that green tea polyphenol EGCG causes a G0/G1-phase cell cycle arrest and apoptosis of human epidermoid carcinoma (A431) cells. We also demonstrated that these effects of EGCG may be mediated through the inhibition of nuclear factor kappa B that has been associated with cell cycle regulation and cancer. In this study, employing A431 cells, we provide evidence for the involvement of cyclin kinase inhibitor (cki)-cyclin-cyclin-dependent kinase (cdk) machinery during cell cycle deregulation by EGCG. As shown by immunoblot analysis, EGCG treatment of the cells resulted in significant dose- and time-dependent (i) upregulation of the protein expression of WAF1/p21, KIP1/p27, p16 and p18, (ii) downmodulation of the protein expression of cyclin D1, cdk4 and cdk6, but not of cyclin E and cdk2, (iii) inhibition of the kinase activities associated with cyclin E, cyclin D1, cdk2, cdk4 and cdk6. Taken together, our study suggests that EGCG causes an induction of G1-phase ckis, which inhibit the cyclin-cdk complexes operative in G0/G1 phase of the cell cycle thereby causing a G0/G1-phase arrest of the cell cycle, which is an irreversible process ultimately resulting in an apoptotic cell death. We suggest that the naturally occurring agents such as green tea polyphenols which may inhibit cell cycle progression could be developed as potent anticancer agents for the management of cancer.     

Cooperative regulation of the cell division cycle by the protein kinases RAF and AKT

The RAS-activated RAF-->MEK-->extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3'-kinase (PI3'-kinase)-->PDK1-->AKT signaling pathways are believed to cooperate to promote the proliferation of normal cells and the aberrant proliferation of cancer cells. To explore the mechanisms that underlie such cooperation, we have derived cells harboring conditionally active, steroid hormone-regulated forms of RAF and AKT. These cells permit the assessment of the biological and biochemical effects of activation of these protein kinases either alone or in combination with one another. Under conditions where activation of neither RAF nor AKT alone promoted S-phase progression, coactivation of both kinases elicited a robust proliferative response. Moreover, under conditions where high-level activation of RAF induced G(1) cell cycle arrest, activation of AKT bypassed the arrest and promoted S-phase progression. At the level of the cell cycle machinery, RAF and AKT cooperated to induce cyclin D1 and repress p27(Kip1) expression. Repression of p27(Kip1) was accompanied by a dramatic reduction in KIP1 mRNA and was observed in primary mouse embryo fibroblasts derived from mice either lacking SKP2 or expressing a T187A mutated form of p27(Kip1). Consistent with these observations, pharmacological inhibition of MEK or PI3'-kinase inhibited the effects of activated RAS on the expression of p27(Kip1) in NIH 3T3 fibroblasts and in a panel of bona fide human pancreatic cancer cell lines. Furthermore, we demonstrated that AKT activation led to sustained activation of cyclin/cdk2 complexes that occurred concomitantly with the removal of RAF-induced p21(Cip1) from cyclin E/cdk2 complexes. Cumulatively, these data strongly suggest that the RAF-->MEK-->ERK and PI3'K-->PDK-->AKT signaling pathways can cooperate to promote G(0)-->G(1)-->S-phase cell cycle progression in both normal and cancer cells.     

MicroRNA-576-3p Inhibits Proliferation in Bladder Cancer Cells by Targeting Cyclin D1

MicroRNAs (miRNAs) are small, endogenous RNAs that play important gene-regulatory roles by binding to the imperfectly complementary sequences at the 3′-UTR of mRNAs and directing their gene expression. Here, we first discovered that miR-576-3p was down-regulated in human bladder cancer cell lines compared with the non-malignant cell line. To better characterize the role of miR-576-3p in bladder cancer cells, we over-expressed or down-regulated miR-576-3p in bladder cancer cells by transfecting with chemically synthesized mimic or inhibitor. The overexpression of miR-576-3p remarkably inhibited cell proliferation via G1-phase arrest, and decreased both mRNA and protein levels of cyclin D1 which played a key role in G1/S phase transition. The knock-down of miR-576-3p significantly promoted the proliferation of bladder cancer cells by accelerating the progression of cell cycle and increased the expression of cyclin D1. Moreover, the dual-luciferase reporter assays indicated that miR-576-3p could directly target cyclin D1 through binding its 3′-UTR. All the results demonstrated that miR-576-3p might be a novel suppressor of bladder cancer cell proliferation through targeting cyclin D1.     

Activation of cyclin-dependent kinases by Myc mediates induction of cyclin A, but not apoptosis.

The activation of conditional alleles of Myc induces both cell proliferation and apoptosis in serum-deprived RAT1 fibroblasts. Entry into S phase and apoptosis are both preceded by increased levels of cyclin E- and cyclin D1-dependent kinase activities. To assess which, if any, cellular responses to Myc depend on active cyclin-dependent kinases (cdks), we have microinjected expression plasmids encoding the cdk inhibitors p16, p21 or p27, and have used a specific inhibitor of cdk2, roscovitine. Expression of cyclin A, which starts late in G1 phase, served as a marker for cell cycle progression. Our data show that active G1 cyclin/cdk complexes are both necessary and sufficient for induction of cyclin A by Myc. In contrast, neither microinjection of cdk inhibitors nor chemical inhibition of cdk2 affected the ability of Myc to induce apoptosis in serum-starved cells. Further, in isoleucine-deprived cells, Myc induces apoptosis without altering cdk activity. We conclude that Myc acts upstream of cdks in stimulating cell proliferation and also that activation of cdks and induction of apoptosis are largely independent events that occur in response to induction of Myc.     

NA22598, a Novel Antitumor Compound, Reduces Cyclin D1 Levels, Arrests Cell Cycle at G1 Phase, and Inhibits Anchorage-Independent Growth of Human Tumor Cells

NA22598, a novel antitumor compound isolated from a microbial cultured broth, inhibited the growth of human colon cancer DLD-1 cells in suspension cultures (anchorage-independent growth) severalfold more strongly than in substratum-attached monolayer cultures. It arrested the cell cycle progression at early G1 phase under both these culture conditions. Rb phosphorylation, cyclin D1 expression, and cdk2 activation in G1 progression were all inhibited by NA22598, but the amounts of cdk2 and p27 were not affected. Among these effects the inhibition of cyclin D1 expression was most prominent, and NA22598 was found to inhibit the synthesis of cyclin D1 without affecting mRNA expression or protein degradation. p27 binding to cdk2 was more markedly increased in suspension cultures than in attached cultures by NA22598, but the compound had no effect on total p27. Apparently, the decrease of cyclin D1 induced redistribution of p27 from the cyclin D1/cdk4 to the cyclin E/cdk2 complexes during G1 phase in the suspension cultures. Because p27 is upregulated during suspension culture, a greater amount of it was associated with cyclin E/cdk2, thus producing greater growth inhibition. An agent, like NA22598, which induces the downregulation of cyclin D1 might offer a new anticancer strategy.