Geraniol and beta-ionone inhibit proliferation, cell cycle progression, and cyclin-dependent kinase 2 activity in MCF-7 breast cancer cells independent of effects on HMG-CoA reductase activity

3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase catalyzes the formation of mevalonate, a precursor of cholesterol that is also required for cell proliferation. Mevalonate depletion results in a G1 phase cell cycle arrest that is mediated in part by impaired activity of cyclin-dependent kinase (CDK) 2, and decreased expression of positive regulators of G1 to S phase progression. Inhibition of mevalonate synthesis may, therefore, be a useful strategy to impair the growth of malignant cells. Plant isoprenoids, including beta-ionone and geraniol, have previously been shown to inhibit rodent mammary tumor development, and rodent and avian hepatic HMG-CoA reductase activity. We hypothesized that the putative anti-proliferative and cell cycle inhibitory effects of beta-ionone and geraniol on MCF-7 human breast cancer cells in culture are mediated by mevalonate depletion resulting from inhibition of HMG-CoA reductase activity. Flow cytometric analysis showed a G1 arrest in isoprenoid-treated MCF-7 cells, and also a G2/M arrest at higher concentrations of isoprenoids. These compounds minimally affected the growth of MCF-10F normal breast epithelial cells. Both beta-ionone and geraniol inhibited CDK 2 activity and dose-dependently decreased the expression of cyclins D1, E, and A, and CDK 2 and 4, without changing the expression of p21cip1 or p27kip1. Although both beta-ionone and geraniol also inhibited MCF-7 proliferation, only geraniol inhibited HMG-CoA reductase activity. While these effects were significantly correlated (r2=0.89, P <0.01), they were not causally related, since exogenous mevalonate did not restore growth in geraniol-inhibited cells. These findings indicate that mechanisms other than impaired mevalonate synthesis mediate the anti-proliferative and cell cycle regulatory effects of beta-ionone and geraniol in human breast cancer cells.     

Comparison of the efficacy of 7-hydroxystaurosporine (UCN-01) and other staurosporine analogs to abrogate cisplatin-induced cell cycle arrest in human breast cancer cell lines

DNA-damaging agents such as cisplatin arrest cell cycle progression at either the G1, S, or G2 phase, although the G1 arrest is seen only in cells expressing the wild-type p53 tumor suppressor protein. Caffeine has been shown to abrogate the S and G2 arrest in p53-defective cells and to enhance cytotoxicity, but at concentrations too toxic to administer to humans. We have reported that 7-hydroxystaurosporine (UCN-01) also overcomes S and G2 phase arrest and enhances the cytotoxicity of cisplatin. We show here that UCN-01 at non-cytotoxic concentrations abrogated S and G2 arrest induced by cisplatin in two p53-defective human breast cancer cell lines. UCN-01 pushed the cells through S phase and mitosis, with subsequent apoptosis. Inhibition of mitosis with nocodazole reduced the apoptosis induced by UCN-01 plus cisplatin. Seven staurosporine analogs were compared for their ability to abrogate cell cycle arrest. Staurosporine was as effective as UCN-01 at abrogating S and G2 arrest, but the concentrations required were cytotoxic. K252a abrogated S phase arrest but failed to abrogate G2 arrest because alone it induced G2 arrest. Hence, K252a did not enhance cisplatin-induced cytotoxicity because it failed to push the cells through a lethal mitosis. None of the other analogs influenced cell cycle progression at the concentrations tested. Accordingly, UCN-01 was the only analog that overcame cell cycle arrest and enhanced the cytotoxicity of cisplatin while exhibiting no cytotoxicity of its own. Hence, UCN-01 remains the most promising candidate for testing clinically in combination with cisplatin.     

Chemical and biological profile of dual Cdk1 and Cdk2 inhibitors

The importance of Cdks in cell cycle regulation, their interaction with oncogenes and tumor suppressors, and their frequent deregulation in human tumors, has encouraged an active search for agents capable of perturbing the function of Cdks. In our laboratories, a variety of selective and potent low molecular weight inhibitors directed against the ATP binding sites of the Cdk1, Cdk2 have been developed. Extensive biological profiling of two distinct classes of Cdk inhibitors - the phenylamino pyrimidines (PAPs) and trisubstituted purines has revealed distinct differences in their cellular effects in normal cells compared to tumor cells. Due to their intact G1/S checkpoints, normal cells are shown to be reversibly blocked by these Cdk inhibitors in either the G1/S-phase or at the G2/M boarder. In transformed cells these control points are either absent or defective and treatment with the compounds resulted in pronounced proliferation block at the G2/M transition. Furthermore, there is strong evidence that this G2/M arrest is less well tolerated by the cells and consequently, they undergo apoptotic cell death. Finally, these dual Cdk1/ Cdk2 inhibitors are also found to be significantly more active on proliferating cells compared to quiescent cells reflecting their specific activity. Despite these encouraging results demonstrating a distinct outcome after treatment with such dual Cdk inhibitors in normal compared to de-regulated tumor cells, it remains to be determined whether a comparable therapeutic window might be observed in vivo experiments. Furthermore the intracellular kinase selectivity of inhibitors which are putatively selective in vitro remains a complicating feature that is only recently begun to be addressed by affinity chromatography and phosphoproteomics techniques. Once efficacy can be demonstrated in animal models at well-tolerated doses, there will be strong evidence for the development of cell cycle antagonists for cancer therapy.     

The role of Krs1 in cell cycle arrest

The Krs1 (kinase responsive to stress 1) kinase has been proposed to mediate signals initiated by various forms of cellular or environmental stress for the process of growth arrest and apoptosis. The functional role of Krs1 in cell growth arrest may involve a constant, stress-independent proteolytic modification of the full-length kinase p63(Krs1), via caspase-like activity, to produce kinase-active kinase fragment, p33(Krs1). Induction of the kinase activity of p33(Krs1) is closely correlated with cell growth arrest in the G(0)/G(1) phase due to serum starvation, contact inhibition or growth-arresting agents. Deactivation of p33(Krs1) is closely associated with cell entry into the cell cycle. In response to stress shock the conversion of p63(Krs1) to p33(Krs1) is enhanced, and the kinase activity of p33(Krs1) is additionally increased in quiescent cells that undergo apoptosis. Both mechanisms of proteolytic modification and protein phosphorylation are involved in a complex control of the kinase activity and function of p33(Krs1) in response to environmental changes leading to cell growth arrest in quiescence or quiescence-related apoptosis. Numerous studies have shown that signaling pathways in malignantly transformed cells are regulated differently from their counterpart pathways in normal counterpart cells in response to stress shock or anticancer agents. It is desirable to develop one type of anticancer agent to selectively growth-arrest normal counterpart cells in G(0)/G(1) phase of the cell cycle through the control of Krs1-involved signaling pathways. The success of arresting normal cells in a quiescent state will provide access for other types of anticancer treatments to induce cell death of cancerous cells, which are resistant to growth arrest in other phases of the cell cycle.     

Growth inhibition by the farnesyltransferase inhibitor FTI-277 involves Bcl-2 expression and defective association with Raf-1 in liver cancer cell lines

Farnesyltransferase inhibitors (FTIs) block the growth of tumor cells in vitro and in vivo with minimal toxicity toward normal cells. In general, inhibition of protein farnesylation results in G0/G1 cell cycle block, G2/M cell cycle arrest, or has no effect on cell cycle progression. One aspect of FTI biology that is poorly understood is the ability of these drugs to induce cancer cell growth arrest at the G2/M phase of cell cycle. In the present study, we investigated the effects of the farnesyltransferase inhibitor FTI-277 on two human liver cancer cell lines, HepG2 and Huh7. Treatment of these cells with FTI-277 inhibited Ras farnesylation in a dose-dependent manner. Both HepG2 and Huh7 cell growth was inhibited by FTI-277 and cells accumulated at the G2/M phase of the cell cycle. In HepG2 and Huh7 cells, FTI-277 induced an up-regulation of the cyclin-dependent kinase inhibitor p27(Kip1) without affecting the cellular levels of p53 and p21(Waf1). This event correlated with reduced activity of the cyclin-dependent kinase 2 and cyclin-dependent kinase 1. Moreover, increased expression of Bcl-2 protein was observed in HepG2 and Huh7 cells treated with FTI-277, and this was coincidental with reduced association between Raf-1 and Bcl-2. Finally, transient transfection of a dominant-negative Ras allele induced Bcl-2 expression and reduced Bcl-2/Raf-1 association demonstrating a requirement for Ras. Taken together, these findings show that increased expression of p27(Kip1) and Bcl-2 is concomitant with altered association between Ras, Raf-1 and Bcl-2 and suggest that this is responsible for the growth-inhibitory properties of FTI-277.     

Mechanism of cell proliferation--cell cycle, oncogenes, and senescence

Cell proliferation is regulated through a transition between the G0 phase and cell cycle. We isolated a mammalian temperature-sensitive mutant cell line defective in the function from the G0 phase to cell cycle. Senescent human somatic cells fail to enter into the cell cycle from the G0 phase with stimulation by any growth factor. Telomere shortening was found to be a cause of cellular senescence, and reexpression of telomerase immortalized human somatic cells. Immortalized human somatic cells showed normal phenotypes and were useful not only for basic research but also for clinical and applied fields. The importance of p53 and p21 activation/induction i now well accepted in the signal transduction process from telomere shortening to growth arrest, but the precise mechanism is largely unknown as yet. We found that the MAP kinase cascade and histone acetylase have an important role in the signaling process to express p21. Tumor tissues and cells were found to have strong telomerase activity, while most normal somatic human tissues showed very weak or no activity. Telomerase activity was shown to be a good marker for early tumor diagnosis because significant telomerase activity was detected in very early tumors or even in some precancerous tissues compared with adjacent normal tissues. Telomere/telomerase is a candidate target for cancer chemotherapeutics, and an agent that abrogated telomere functions was found to kill tumor cells effectively by inducing apoptosis whereas it showed no effect on the viability of normal cells.     

Natural flavonoids targeting deregulated cell cycle progression in cancer cells

The prolonged duration requiring alteration of multi-genetic and epigenetic molecular events for cancer development provides a strong rationale for cancer prevention, which is developing as a potential strategy to arrest or reverse carcinogenic changes before the appearance of the malignant disease. Cell cycle progression is an important biological event having controlled regulation in normal cells, which almost universally becomes aberrant or deregulated in transformed and neoplastic cells. In this regard, targeting deregulated cell cycle progression and its modulation by various natural and synthetic agents are gaining widespread attention in recent years to control the unchecked growth and proliferation in cancer cells. In fact, a vast number of experimental studies convincingly show that many phytochemicals halt uncontrolled cell cycle progression in cancer cells. Among these phytochemicals, natural flavonoids have been identified as a one of the major classes of natural anticancer agents exerting antineoplastic activity via cell cycle arrest as a major mechanism in various types of cancer cells. This review is focused at the modulatory effects of natural flavonoids on cell cycle regulators including cyclin-dependent kinases and their inhibitors, cyclins, p53, retinoblastoma family of proteins, E2Fs, check-point kinases, ATM/ATR and survivin controlling G1/S and G2/M check-point transitions in cell cycle progression, and discusses how these molecular changes could contribute to the antineoplastic effects of natural flavonoids.     

Cytostatic synergism between bromodeoxyuridine, bleomycin, cisplatin and chlorambucil demonstrated by a sensitive cell kinetic assay.

Bromodeoxyuridine/Hoechst flow cytometry was used to analyse the interference of common cytostatic agents with cell activation and cell cycle progression of human B-cell lines. Bleomycin impaired both cell activation and G2 transit, the latter effect being oxygen dependent. The DNA alkylating agents cyclophosphamide, chlorambucil and mitomycin C caused G2 arrest, whereas cisplatin arrested cells in both the S and G2 phase of the cell cycle. Vinblastin interfered with mitosis, but in addition arrested cells in all phases of the cell cycle. The growth inhibitory action of bleomycin, cisplatin and chlorambucil was dependent upon the bromodeoxyuridine (BrdU) concentration in the culture medium. No interaction was found between BrdU and cyclophosphamide, mitomycin C and vinblastin. The cell cycle kinetic mechanism of the interaction between BrdU and bleomycin, cisplatin and chlorambucil was a potentiation of the G2 arrest. In conclusion, BrdU may be useful in clinical chemotherapy as a chemosensitizer for selected cytostatic agents.     

The combination of sulfinosine and 8-Cl-cAMP induces synergistic cell growth inhibition of the human neuroblastoma cell line in vitro

Summary To identify purine analogs that could be effective in treating neuroblastomas, we tested the anticancer properties of sulfinosine, 8-Cl-cAMP and 8-Cl-adenosine in the SK-N-SH cell line. First we examined the effects of these three agents on cell growth inhibition and cell viability by the BrdU and Sulforhodamine B assay. Treatment of SK-N-SH cells with increasing concentrations of these compounds led to a significant inhibition of cell proliferation and decrease of cell viability in a time- and dose-dependant manner at micromolar concentration (<10 μM). Treatment with a combination of sulfinosine and 8-Cl-cAMP resulted in synergistic effects on growth inhibition, cell cycle arrest and induction of apoptosis. Flow-cytometric analysis showed that 8-Cl-cAMP arrested the cells in the G0/G1 phase and sulfinosine blocked cell cycle progression at the G2/M stage, in contrast to the combined effects of both agents that did not arrest growth at any particular phase of the cell cycle. Further analysis of apoptosis induction demonstrated an increase from 17 to 24% of both early and late apoptotic cells and a very low percentage of necrotic cells. These results indicate that apoptosis was the predominant type of cell death after treatment of SK-N-SH cells with both substances, as well as with their combinations.     

Permanent cell cycle arrest in asynchronously proliferating normal human fibroblasts treated with doxorubicin or etoposide but not camptothecin.

Damage to DNA has been implicated in the induction of permanent cell cycle arrest or premature senescence in normal human fibroblasts. We tested the ability of a group of cancer chemotherapeutic agents or related compounds, which can cause DNA double-strand breaks (DSBs) directly or indirectly, to induce a permanent cell cycle arrest in normal proliferating fibroblasts. A brief treatment with etoposide, doxorubicin, cisplatin, or phleomycin D1 induced a block to S phase entry sustained through 15 days. Lower levels of these drugs did not induce appreciable levels of transient cell cycle arrest. Higher concentrations caused cell death that lacked the DNA degradation characteristic of apoptosis. Camptothecin, an agent that causes DNA single-strand breaks, which are converted to DSBs during S phase, was able to induce an efficient, but only transient, cell cycle arrest in these normal cells. The cells did not enter S phase until after removal of the camptothecin. These findings support the idea that permanent cell cycle arrest and cell death are typical reactions of these normal cells to drugs that can cause DSBs. In addition, we report data consistent with the concept that both actinomycin D and doxorubicin are sequestered by cells and slowly released in active form. This is consistent with the observation that both these drugs bind reversibly to intracellular components.     

Synthesis and biological evaluation of 1-benzylidene-3,4-dihydronaphthalen-2-one as a new class of microtubule-targeting agents

A series of 1-benzylidene-3,4-dihydronaphthalen-2-one derivatives were designed and synthesized, and their biological activities in vitro and in vivo were evaluated. The results showed a number of the title compounds exhibiting potent nanomolar activity in several human cancer cell lines. Of these, compound 22b showed the strongest inhibitory activity against human CEM, MDA-MBA-435, and K562 cells (IC(50) = 1 nM), displayed in vitro inhibition of tubulin polymerization (IC(50) = 3.93 μM), and significantly induced cell cycle arrest in G2/M phase. In addition, compound 22b could inhibit the tumor growth in colon nude mouse xenograft tumor model significantly and seemed safer than CA-4 when achieving a similar tumor suppression. This study provided a new molecular scaffold for the further development of antitumor agents that target tubulin.     

Microtubule damaging agents induce apoptosis in HL 60 cells and G2/M cell cycle arrest in HT 29 cells

Microtubule damaging agents (such as paclitaxel and nocodazole (ND)) have been used in the clinical cancer chemotherapy. However, the molecular mechanisms of these agents in the induction of anti-cancer activity are still unclear. In the present study, we demonstrated that 0.2 microM podophyllotoxin (PDP) induced the occurrence of apoptosis in human leukemic (HL 60) cells and cell cycle arrest at the G2/M phase in HT 29 cells. Our results suggest that the PDP-induced G2/M arrest in HT 29 cells was through the intracellular events including (a) inhibition of normal mitotic spindle formation, (b) elevation of cyclin B1/cdc2 kinase activity, (c) concomitant increases in cdc 25 A phosphatase and cdk 7 kinase activity, and (d) down-regulation of the wee-1 protein expression. On the other hand, activations of the caspases 3, 8, and 9, Bcl-2 hyper-phosphorylation, and increased leakage of cytochrome c from mitochondria into cytosolic fraction were detected in the PDP-treated HL 60 cells. These listed intracellular events were interpreted to lead to the apoptosis observed in PDP-treated HL 60 cells. We further demonstrated that activation of c-jun N-terminal kinase (JNK) signaling pathway may play an important role in the PDP-induced Bcl-2 phosphorylation and apoptosis in HL 60 cells as evidenced by the JNK specific anti-sense oligonucleotide experiment. Our results demonstrated that the occurrence of apoptosis or G2/M cell cycle arrest induced by microtubule damaging agents in different cancer cells was through independent mechanisms. The results from the present study highlight the molecular mechanisms underlying of the PDP-induced anti-cancer activity.     

Effect of tetrandrine combined with epirubicin on the growth of human breast carcinoma multidrug resistance cell line

Multidrug resistance presents a serious problem in cancer chemotherapy. Recent studies have shown that the multidrug resistance of tumor cells can be reversed by tetrandrine by potentiating the cytotoxicity of chemotherapeutic agents. However, whether tetrandrine has such potentiating effect on epirubicin has not been reported. Thus, the combined effect of tetrandrine and epirubicin on the growth of human breast carcinoma multidrug-resistant MCF-7/ADM cells was studied in the present study. It was shown that tetrandrine significantly potentiated the cytotoxicity of epirubicin. To examine the mechanism of the combined effect of tetrandrine and epirubicin on MCF-7/ADM cell growth, cell cycle progression was evaluated by using flow cytometry. The combined use of tetrandrine and epirubicin caused an accumulation of cells at G(2)/M phase, accompanied with a concomitant decrement of cell number at G(0)/G(1) phase. The present study demonstrated for the first time that tetrandrine potentiated the cytotoxcity of epirubicin on MCF-7/ADM cells. Cell cycle arrest at G(2)/M phase may contribute to the combined effect of tetrandrine and epirubicin.     

Biphasic effect of daidzein on cell growth of human colon cancer cells.

Colorectal cancer is one of the most common cancers in the world, poorly responding to available chemotherapeutic agents. To investigate whether natural molecules can inhibit colon cancer progression, we investigated a principle phytoestrogen found in soybean known as daidzein, and determined its effects on the human colon cancer cell line LoVo. LoVo cells were treated with 0.1, 1, 5, 10, 50 and 100 microM daidzein for 2, 3, 4 or 5 d. The results indicated that daidzein stimulated the growth of LoVo cells at 0.1 and 1 microM whereas at higher concentrations (10, 50 and 100 microM) cell growth was inhibited in a dose-dependent manner. Treatment of daidzein at 10, 50 and 100 microM resulted in cell cycle arrest at G0/G1 phase, DNA fragmentation and increases in caspase-3 activity. There were no changes in alkaline phosphatase activity (ALP), an indicator of cell differentiation, upon treatment with daidzein when compared to controls. These results indicate that daidzein has a biphasic effect on LoVo cell growth and its tumor suppressive effect is by means of cell cycle arrest and apoptosis but not through cell differentiation.     

Induction of senescent-like growth arrest as a new target in anticancer treatment

Replicative senescence is a programmed cellular response in normal cells, the induction of which depends on the accumulated number of cell divisions. Unlike cells undergoing apoptosis, senescent cells have a large and flat morphology, express acidic beta-galactosidase (beta-gal) and show a permanent cell cycle G(1) phase arrest. Recently, senescent-like growth arrest has been observed in many types of tumor cell lines after exposure to certain chemotherapeutic drugs. These senescent-like cancer cells show similar morphology, growth arrest and beta-gal expression to normal cells undergoing replicative senescence. However, unlike replicative senescence during the aging process, the chemodrug-induced senescent-like growth arrest is independent of cell cycle distribution, telomere length or cell cycle inhibitors. These observations suggest that induction of senescent-like response may provide a novel target leading to permanent growth arrest in cancer cells. So far, cell lines derived from more than 14 types of cancers have shown senescent-like growth arrest by either introduction of tumor suppressor genes or treatment with chemotherapeutic drugs. In addition, the drug-induced beta-gal expression has been correlated with cancer cells undergoing terminal senescent-like growth arrest, which provides a possible marker for this process. This review will describe the evidence on senescent-like growth arrest in human cancer cells and the molecular changes that differ between chemodrug-induced senescent-like growth arrest and apoptosis. In addition, the possible factors and mechanisms involved in this process are also discussed. Finally, the implications on how senescent-like growth arrest might be exploited as a possible new target for anti-cancer drugs are addressed.     

Difference of cell cycle arrests induced by lidamycin in human breast cancer cells

Lidamycin (LDM) is a member of the enediyne antibiotic family. It is undergoing phase I clinical trials in China as a potential chemotherapeutic agent. In the present study, we investigated the mechanism by which LDM induced cell cycle arrest in human breast cancer cells. The results showed that LDM induced G1 arrest in p53 wild-type MCF-7 cells at low concentrations, and caused both G1 and G2/M arrests at higher concentrations. In contrast, LDM induced only G2/M arrest in p53-mutant MCF-7/DOX cells. Western blotting analysis indicated that LDM-induced G1 and G2/M arrests in MCF-7 cells were associated with an increase of p53 and p21, and a decrease of phosphorylated retinoblastoma tumor suppressor protein, cyclin-dependent kinase (Cdk), Cdc2 and cyclin B1 protein levels. However, LDM-induced G2/M arrest in MCF-7/DOX cells was correlated with the reduction of cyclin B1 expression. Further study indicated that the downregulation of cyclin B1 by LDM in MCF-7 cells was associated with decreasing cyclin B1 mRNA levels and promoting protein degradation, whereas it was only due to inducing cyclin B1 protein degradation in MCF-7/DOX cells. In addition, activation of checkpoint kinases Chk1 or Chk2 maybe contributed to LDM-induced cell cycle arrest. Taken together, we provide the first evidence that LDM induces different cell cycle arrests in human breast cancer cells, which are dependent on drug concentration and p53 status. These findings are helpful in understanding the molecular anti-cancer mechanisms of LDM and support its clinical trials.     

Hexachlorobenzene induces TGF-β1 expression,which is a regulator of p27 and cyclin D1 modifications

Hexachlorobenzene (HCB) is an organochlorine pesticide widely distributed in the environment. In this study we have demonstrated that HCB induced loss of cell viability and alterations in cell cycle regulation in FRTL-5 rat thyroid cells. Analysis of cell cycle distribution by flow cytometric analysis demonstrated that HCB induced cell cycle arrest at G2/M and at G0/G1 phase, inhibiting cell cycle progression at the G1/S phase, after 24 h and 72 h of treatment.     

The in-vitro antiproliferative effect of PRI-2191 and imatinib applied in combined treatment with cisplatin, idarubicin, or docetaxel on human leukemia cells

Imatinib mesylate (Gleevec, STI571) is a specific inhibitor of the Bcr/Abl fusion tyrosine kinase that exhibits potent antileukemic effects in chronic myelogenous leukemia. Bcr/Abl-positive K562 and Bcr/Abl-negative HL-60 human leukemia cells were used to investigate the effect of PRI-2191, a calcitriol analog, on the biological effects of imatinib combined with other anticancer drugs. The results show that PRI-2191 enhances the antiproliferative effect of imatinib on HL-60 cells. When these two agents together are applied with either docetaxel or cisplatin, but not with idarubicin, the antiproliferative effect could still be enhanced. Moreover, when the interaction between the chemotherapy agents was antagonistic or additive, PRI-2191 could even shift it to synergism. This effect correlated with an accumulation of HL-60 cells in the G0/G1 phase of the cell cycle and a decrease in the percentage of cells in the G2/M and S stage in the ternary combinations used. PRI-2191 did not influence apoptosis induced by imatinib alone or in ternary combinations with all the chemotherapy agents used. These results may suggest that the stronger antiproliferative effect of the combined treatment with PRI-2191 on HL-60 cells is related to cell cycle arrest rather than to the induction of apoptosis.