Enhanced Sensitivity of G1 Arrested Human Cancer Cells Suggests a Novel Therapeutic Strategy Using a Combination of Simvastatin and TRAIL

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) preferentially induces apoptosis in tumor cells over normal cells. To study the relationship between cell cycle progression and TRAIL-induced apoptosis, SW480 colon cancer and H460 lung cancer cell lines were examined for their sensitivity to TRAIL after arrest in different cell cycle phases. Cells were synchronized in G0/G1, S, and G2/M phase by serum starvation, aphidicolin, or nocodazole treatment, respectively. We found that arrest of cells in G0/G1 phase confers significantly higher susceptibility to TRAIL-induced apoptosis as compared to cells in late G1, S, or G2/M phase. To determine if cell cycle phase could be harnessed for therapeutic gain in the presence of TRAIL, we used the HMG-CoA reductase inhibitor, Simvastatin and lovastatin, to enrich a cancer cell population in G0/G1. Both simvastatin and lovastatin significantly augmented TRAIL-induced apoptosis in tumor cells, but not in normal keratinocytes. The results indicate that TRAIL, in combination with a HMG-CoA reductase inhibitor, may have therapeutic potential in the treatment of human cancer.

Key Words

TRAIL, Synchronization, Simvastatin, Cancer Therapy, Lovastatin, Cell Cycle, Apoptosis     

Dose-dependent effects of lovastatin on cell cycle progression. Distinct requirement of cholesterol and non-sterol mevalonate derivatives

The mevalonate pathway is tightly linked to cell proliferation. The aim of the present study is to determine the relationship between the inhibition of this pathway by lovastatin and the cell cycle. HL-60 and MOLT-4 human cell lines were cultured in a cholesterol-free medium and treated with increasing concentrations of lovastatin, and their effects on cell proliferation and the cell cycle were analyzed. Lovastatin was much more efficient in inhibiting cholesterol biosynthesis than protein prenylation. As a result of this, lovastatin blocked cell proliferation at any concentration used, but its effects on cell cycle distribution varied. At relatively low lovastatin concentrations (less than 10 microM), cells accumulated preferentially in G(2) phase, an effect which was both prevented and reversed by low-density lipoprotein cholesterol. At higher concentrations (50 microM), the cell cycle was also arrested at G(1) phase. In cells treated with lovastatin, those arrested at G(1) progressed through S upon mevalonate provision, whereas cholesterol supply allowed cells arrested at G(2) to traverse M phase. These results demonstrate the distinct roles of mevalonate, or its non-sterol derivatives, and cholesterol in cell cycle progression, both being required for normal cell cycling.     

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.     

Lovastatin‐mediated MCF‐7 cancer cell death involves LKB1‐AMPK‐p38MAPK‐p53‐survivin signalling cascade

There is increasing evidence that statins, which are widely used in lowering serum cholesterol and the incidence of cardiovascular diseases, also exhibits anti‐tumour properties. The underlying mechanisms by which statins‐induced cancer cell death, however, remain incompletely understood. In this study, we explored the anti‐tumour mechanisms of a lipophilic statin, lovastatin, in MCF‐7 breast cancer cells. Lovastatin inhibited cell proliferation and induced cell apoptosis. Lovastatin caused p21 elevation while reduced cyclin D1 and survivin levels. Lovastatin also increased p53 phosphorylation, acetylation and its reporter activities. Results from chromatin immunoprecipitation analysis showed that p53 binding to the survivin promoter region was increased, while Sp1 binding to the region was decreased, in MCF‐7 cells after lovastatin exposure. These actions were associated with liver kinase B1 (LKB1), AMP‐activated protein kinase (AMPK) and p38 mitogen‐activated protein kinase (p38MAPK) activation. Lovastatin's enhancing effects on p53 activation, p21 elevation and survivin reduction were significantly reduced in the presence of p38MAPK signalling inhibitor. Furthermore, LKB1‐AMPK signalling blockade abrogated lovastatin‐induced p38MAPK and p53 phosphorylation. Together these results suggest that lovastatin may activate LKB1‐AMPK‐p38MAPK‐p53‐survivin cascade to cause MCF‐7 cell death. The present study establishes, at least in part, the signalling cascade by which lovastatin induces breast cancer cell death.     

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.     

Molecular pathway for (-)-epigallocatechin-3-gallate-induced cell cycle arrest and apoptosis of human prostate carcinoma cells

Epigallocatechin-3-gallate (EGCG), the major polyphenolic constituent present in green tea, is a promising chemopreventive agent. We recently showed that green tea polyphenols exert remarkable preventive effects against prostate cancer in a mouse model and many of these effects are mediated by the ability of polyphenols to induce apoptosis in cancer cells [Proc. Natl. Acad. Sci. USA 98 (2001) 10350]. Earlier, we showed that EGCG causes a G0/G1 phase cell cycle arrest and apoptosis of both androgen-sensitive LNCaP and androgen-insensitive DU145 human prostate carcinoma cells, irrespective of p53 status [Toxicol. Appl. Pharmacol. 164 (2000) 82]. Here, we provide molecular understanding of this effect. We tested a hypothesis that EGCG-mediated cell cycle dysregulation and apoptosis is mediated via modulation of cyclin kinase inhibitor (cki)-cyclin-cyclin-dependent kinase (cdk) machinery. As shown by immunoblot analysis, EGCG treatment of LNCaP and DU145 cells resulted in significant dose- and time-dependent (i) upregulation of the protein expression of WAF1/p21, KIP1/p27, INK4a/p16, and INK4c/p18, (ii) down-modulation of the protein expression of cyclin D1, cyclin E, cdk2, cdk4, and cdk6, but not of cyclin D2, (iii) increase in the binding of cyclin D1 toward WAF1/p21 and KIP1/p27, and (iv) decrease in the binding of cyclin E toward cdk2. Taken together, our results suggest that EGCG causes an induction of G1 phase ckis, which inhibits the cyclin-cdk complexes operative in the G0/G1 phase of the cell cycle, thereby causing an arrest, which may be an irreversible process ultimately leading to apoptotic cell death. This is the first systematic study showing the involvement of each component of cdk inhibitor-cyclin-cdk machinery during cell cycle arrest and apoptosis of human prostate carcinoma cells by EGCG.     

Assessment of lovastatin application as tool in probing cytokinin-mediated cell cycle regulation

Lovastatin, a potent inhibitor of the mevalonate pathway, has been used in plant cell cycle studies to eliminate the cytosolic cytokinin biosynthesis. However, several implications can blur the results, as cytokinins may be alternatively formed from isopentenylpyrophosphate produced by the plastid 1-deoxy-xylulose 5-phosphate pathway and because the endogenous cytokinin levels oscillate considerably in the course of a cell cycle. In the work presented here, short- and long-term effects of lovastatin on suspension- cultured Nicotiana tabacum (L.) BY-2 cells were differentiated. The short-term experiments revealed a fast action of lovastatin, resulting in a significantly, though not completely, decreased content of endogenous cytokinins that became visible already after 10 min and was most pronounced after 30 min. But the impact of lovastatin on cell cycle progression depended also on the phase of the cell cycle at which it was administered. Lowering of the cytokinin level during the early S phase, when the endogenous cytokinin levels increased, delayed the S/G2 transition, whereas the same treatment in the late S phase, when the cellular cytokinin concentrations had already started to decrease, promoted it. Incubation periods longer than 48 h resulted in about 50% loss of viable of the cells and also in a reduced capability of division of the survivors. These cells later on resumed cell division. A second treatment with lovastatin of that culture again killed about 50% of the cells, but the surviving cells showed faster re-growth. In conclusion, lovastatin appears as a useful inhibitor of cytokinin biosynthesis in short-term studies, but its use in long-term experiments may create complex effects and therefore requires substantial caution.     

An overview on the biological activity and anti-cancer mechanism of lovastatin

Lovastatin, a secondary metabolite isolated from fungi, is often used as a representative drug to reduce blood lipid concentration and treat hypercholesterolemia. Its structure is similar to that of HMG-CoA. Lovastatin inhibits the binding of the substrate to HMG-CoA reductase, and strongly competes with HMG-CoA reductase (HMGR), thereby exerting a hypolipidemic effect. Further, its safety has been confirmed in vivo and in vitro. Lovastatin also has anti-inflammatory, anti-cancer, and neuroprotective effects. Therefore, the biological activity of lovastatin, especially its anti-cancer effect, has garnered research attention. Several in vitro studies have confirmed that lovastatin has a significant inhibitory effect on cancer cell viability in a variety of cancers (such as breast, liver, cervical, lung, and colon cancer). At the same time, lovastatin can also increase the sensitivity of some types of cancer cells to chemotherapeutic drugs and strengthen their therapeutic effect. Lovastatin inhibits cell proliferation and regulates cancer cell signaling pathways, thereby inducing apoptosis and cell cycle arrest. This article reviews the structure, biosynthetic pathways, and applications of lovastatin, focusing on the anti-cancer effects and mechanisms of action.     

Effects of Cyclic AMP on Components of the Cell Cycle Machinery Regulating DNA Synthesis in Cultured Astrocytes

Cyclic AMP is a second messenger for various hormones that inhibits cell multiplication and DNA synthesis in cultured astrocytes. We examined the effects of increasing intracellular cyclic AMP on the catalytic (cdks) and regulatory (cyclins and ckis) components of cyclin-dependent protein kinases, which regulate progression of the cell cycle before completion of DNA synthesis, in primary cultured astrocytes and in an astrocytic cell line C.LT.T.1.1. The amount of cdk4 changed little during the cell cycle and was not affected by cyclic AMP. There was little cdk1 and cdk2 in quiescent cells, and their expression increased during the G1-S phases. Cyclic AMP strongly inhibited cdk1 and cdk2 expression. Transforming growth factor beta also inhibited cdk1 expression in primary astrocytes. Cyclic AMP did not affect the two ckis p27KIP1 and p21CIP1. There was little cyclin D1 in quiescent cells, but it increased during the G1 phase and was reduced by cyclic AMP. Cyclin E increased during the G1-S phases and was not affected by cyclic AMP in primary astrocytes. The amount of cyclin A was low in quiescent cells and increased during the G1-S phases. Expression of its mRNA and protein was inhibited by cyclic AMP. The protein kinase activities associated with complexes of cyclins and cdks were increased by growth factors and prevented by cyclic AMP. We conclude that cyclic AMP inhibits progression of the cell cycle in astrocytes at least by preventing the expression of the regulatory subunits, cyclins D1 and A, and catalytic subunits, cdk1 and cdk2, of cyclin-regulated protein kinases. Key Words: Cyclin-dependent protein kinases-Glial cells-Cyclic AMP.     

Ras triggers ataxia-telangiectasia-mutated and Rad-3-related activation and apoptosis through sustained mitogenic signaling

Genetic evidence indicates that Ras plays a critical role in the initiation and progression of human thyroid tumors. Paradoxically, acute expression of activated Ras in normal rat thyroid cells induced deregulated cell cycle progression and apoptosis. We investigated whether cell cycle progression was required for Ras-stimulated apoptosis. Ras increased CDK-2 activity following its introduction into quiescent cells. Apoptotic cells exhibited a sustained increase in CDK-2 activity, accompanied by the loss of CDK-2-associated p27. Blockade of Ras-induced CDK-2 activity and S phase entry via overexpression of p27 inhibited apoptosis. Inactivation of the retinoblastoma protein in quiescent cells through expression of HPV-E7 stimulated cell cycle progression and apoptosis, indicating that deregulated cell cycle progression is sufficient to induce apoptosis. Ras failed to induce G1 phase growth arrest in normal rat thyroid cells. Rather, Ras-expressing thyroid cells progressed into S and G2 phases and evoked a checkpoint response characterized by the activation of ATR. Ras-stimulated ATR activity, as evidenced by Chk1 and p53 phosphorylation, was blocked by p27, suggesting that cell cycle progression triggers checkpoint activation, likely as a consequence of replication stress. These data reveal that Ras is capable of inducing a DNA damage response with characteristics similar to those reported in precancerous lesions. Our findings also suggest that the frequent mutational activation of Ras in thyroid tumors reflects the ability of Ras-expressing cells to bypass checkpoints and evade apoptosis rather than to simply increase proliferative potential.     

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.     

Enhanced sensitivity of G1 arrested human cancer cells suggests a novel therapeutic strategy using a combination of simvastatin and TRAIL

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) preferentially induces apoptosis in tumor cells over normal cells. To study the relationship between cell cycle progression and TRAIL-induced apoptosis, SW480 colon cancer and H460 lung cancer cell lines were examined for their sensitivity to TRAIL after arrest in different cell cycle phases. Cells were synchronized in G0/G1, S, and G2/M phase by serum starvation, aphidicolin, or nocodazole treatment, respectively. We found that arrest of cells in G0/G1 phase confers significantly higher susceptibility to TRAIL-induced apoptosis as compared to cells in late G1, S, or G2/M phase. To determine if cell cycle phase could be harnessed for therapeutic gain in the presence of TRAIL, we used the HMG-CoA reductase inhibitor, Simvastatin and lovastatin, to enrich a cancer cell population in G0/G1. Both simvastatin and lovastatin significantly augmented TRAIL-induced apoptosis in tumor cells, but not in normal keratinocytes. The results indicate that TRAIL, in combination with a HMG-CoA reductase inhibitor, may have therapeutic potential in the treatment of human cancer.     

Inhibition of Cdk1 by Alsterpaullone and Thioflavopiridol Correlates with Increased Transit Time from Mid G2 Through Prophase

Current models suggest that cyclin B1/cdk1 regulates the G2 to M transition and that its activity is maximal during the period from prophase to metaphase in mammalian cells. Although data are lacking, the idea that cyclin B1/cdk1 regulates the transit time from prophase to metaphase is reasonable. Development of small molecule inhibitors of cyclin dependent kinases (cdk’s) as cancer therapeutics presents an opportunity to evaluate the effects of inhibiting cdk’s in asynchronous cell populations. Analysis of cdk1 inhibitors is complicated by their ability to inhibit other cdk’s in vitro at higher concentrations. In this study we measured the effects of two cdk1 inhibitors on S, G2, and M transit for Hela cells and correlated these effects on cyclin B1/cdk1 and cyclin A/cdk2 activities. Dose responses demonstrate that low concentrations of both compounds inhibited the activity of cdk1 but not cdk2 in HeLa cells. The partial loss of cdk1 activity at low doses induced a prophase accumulation during a 3 h period and an increased transit time through mitosis. In addition, both inhibitors lengthened the G2 transit time with progressively greater effect on mid and late G2. High doses of both inhibitors increased the S phase time, which correlated with the inhibition of cdk2 activity. These results suggest that cdk1-cyclin activity is rate limiting for cell cycle progression during a period from mid G2 through prophase.     

Lovastatin-induced up-regulation of the BH3-only protein, Bim, and cell death in glioblastoma cells

The mechanism of lovastatin-induced cell death was examined in three established human glioblastoma cell lines; U87, U251, and U138. Changes in potential modifiers of apoptosis, including Bcl-2 family proteins and MAP kinase targets after such lovastatin treatment, were evaluated. Lovastatin (5 microm) treatment causes extensive cell death in two of the cell lines, U87 and U251; but only minimal in a third, U138. Lovastatin-induced death occurs in correlation with significantly increased levels of the BH3-only protein, Bim. The up-regulation of Bim levels was directly associated with an increased incidence of apoptosis. Lovastatin treatment in U87 cells results in activation of targets of three major mitogen-activating protein kinase cascades including Erk1/2, JNK and p38. Changes in levels of Bim, as well as increase phosphorylation of Erk1/2, c-jun, and p38 are all prevented by co-incubation of lovastatin and the isoprenylation metabolite, geranylgeranyl pyrophosphate. Inhibition of the MAP kinase pathways failed to block the increased expression of Bim expression or cell death. Further elucidation of the mechanisms of lovastatin-induced up-regulation of Bim and apoptosis in glioblastoma cells are important in determining a potential role for lovastatin as a chemotherapy agent.     

TNFalpha-mediated cell death is independent of cdc25A

Tumor necrosis factor (TNFs) have been shown to be synthesized by ovarian carcinomas, and may therefore affect tumor cells in an autocrine manner. Therefore, we investigated the effects of recombinant TNFs on ovarian carcinoma cells N.1 and examined expression of the proto-oncogenes c-myc and cdc25A which are known to play a prominent role in apoptosis. TNFalpha elicited apoptosis in N.1 cells within 72 h which was shown by typical morphological changes, DNA fragmentation and signature type cleavage of poly(ADP-ribose) polymerase into a 89 kDa proteolytic peptide. TNFalpha-induced apoptosis was accompanied by constitutive c-Myc expression, although the mRNA level of phosphatase cdc25A was suppressed within 24 h of TNFalpha treatment and the protein level decreased after 48 h. Cdc25A tyrosine phosphatase is an activator of the cdk2-cyclin E complex which allows for cell cycle progression. As expected, we found TNFalpha-mediated Cdc25A down-regulation to inhibit Cdk2 activity. Cdc25A suppression was related to TNFalpha-induced apoptosis but not to a TNFalpha-induced G0 arrest because cyclin D1 expression was unaffected and the gene gas6 (growth arrest specific 6) was not induced. Arresting cells by treatment with genistein prevented TNFalpha-triggered apoptosis and inhibited c-myc expression. TNFalpha-induced apoptosis is not accompanied by cell cycle arrest which may be due to constitutive c-Myc expression, although Cdc25A and Cdk2 activity is also down-regulated. High c-Myc and low Cdc25A activity might present conflicting signals to the cell cycle machinery which are incompatible with cell survival.     

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.     

Inhibition of cdk1 by alsterpaullone and thioflavopiridol correlates with increased transit time from mid G2 through prophase

Current models suggest that cyclin B1/cdk1 regulates the G2 to M transition and that its activity is maximal during the period from prophase to metaphase in mammalian cells. Although data are lacking, the idea that cyclin B1/cdk1 regulates the transit time from prophase to metaphase is reasonable. Development of small molecule inhibitors of cyclin dependent kinases (cdk's) as cancer therapeutics presents an opportunity to evaluate the effects of inhibiting cdk's in asynchronous cell populations. Analysis of cdk1 inhibitors is complicated by their ability to inhibit other cdk's in vitro at higher concentrations. In this study we measured the effects of two cdk1 inhibitors on S, G2, and M transit for Hela cells and correlated these effects on cyclin B1/cdk1 and cyclin A/cdk2 activities. Dose responses demonstrate that low concentrations of both compounds inhibited the activity of cdk1 but not cdk2 in HeLa cells. The partial loss of cdk1 activity at low doses induced a prophase accumulation during a 3 h period and an increased transit time through mitosis. In addition, both inhibitors lengthened the G2 transit time with progressively greater effect on mid and late G2. High doses of both inhibitors increased the S phase time, which correlated with the inhibition of cdk2 activity. These results suggest that cdk1-cyclin activity is rate limiting for cell cycle progression during a period from mid G2 through prophase.     

Lovastatin inhibits human B lymphoma cell proliferation by reducing intracellular ROS and TRPC6 expression

Clinical evidence suggests that statins reduce cancer incidence and mortality. However, there is lack of in vitro data to show the mechanism by which statins can reduce the malignancies of cancer cells. We used a human B lymphoma Daudi cells as a model and found that lovastatin inhibited, whereas exogenous cholesterol (Cho) stimulated, proliferation cell cycle progression in control Daudi cells, but not in the cells when transient receptor potential canonical 6 (TRPC6) channel was knocked down. Lovastatin decreased, whereas Cho increased, the levels of intracellular reactive oxygen species (ROS) respectively by decreasing or increasing the expression of p47-phox and gp91-phox (NOX2). Reducing intracellular ROS with either a mimetic superoxide dismutase (TEMPOL) or an NADPH oxidase inhibitor (apocynin) inhibited cell proliferation, particularly in Cho-treated cells. The effects of TEMPOL or apocynin were mimicked by inhibition of TRPC6 with SKF-96365. Lovastatin decreased TRPC6 expression and activity via a Cho-dependent mechanism, whereas Cho increased TRPC6 expression and activity via an ROS-dependent mechanism. Consistent with the fact that TRPC6 is a Ca^2 +-permeable channel, lovastatin decreased, but Cho increased, intracellular Ca^2 + also via ROS. These data suggest that lovastatin inhibits malignant B cell proliferation by reducing membrane Cho, intracellular ROS, TRPC6 expression and activity, and intracellular Ca^2 +.