The simple ganglioside GM3 has been shown to have anti-proliferative effects in several in vitro and in vivo cancer models. Although the exogenous ganglioside GM3 has an inhibitory effect on cancer cell proliferation, the exact mechanism by which it prevents cell proliferation remains unclear. Previous studies showed that MDM2 is an oncoprotein that controls tumorigenesis through both p53-dependent and p53-independent mechanisms, and tumor suppressor phosphatase and tensin homolog deleted on chromosome 10 (PTEN), a dual-specificity phosphatase that antagonizes phosphatidylinositol 3-kinase (PI-3K)/AKT signaling, is capable of blocking MDM2 nuclear translocation and destabilizing the MDM2 protein. Results from our current study show that GM3 treatment dramatically increases cyclin-dependent kinase (CDK) inhibitor (CKI) p21WAF1 expression through the accumulation of p53 protein by the PTEN-mediated inhibition of the PI-3K/AKT/MDM2 survival signaling in HCT116 colon cancer cells. Moreover, the data herein clearly show that ganglioside GM3 induces p53-dependent transcriptional activity of p21WAF1, as evidenced by the p21WAF1 promoter-driven luciferase reporter plasmid (full-length p21WAF1 promoter and a construct lacking the p53-binding sites). Additionally, ganglioside GM3 enhances expression of CKI p27kip1 through the PTEN-mediated inhibition of the PI-3K/AKT signaling. Furthermore, the down-regulation of the cyclin E and CDK2 was clearly observed in GM3-treated HCT116 cells, but the down-regulation of cyclin D1 and CDK4 was not. On the contrary, suppression of PTEN levels by RNA interference restores the enhanced expression of p53-dependent p21WAF1 and p53-independent p27kip1 through inactivating the effect of PTEN on PI-3K/AKT signaling modulated by ganglioside GM3. These results suggest that ganglioside GM3-stimulated PTEN expression modulates cell cycle regulatory proteins, thus inhibiting cell growth. We conclude that ganglioside GM3 represents a modulator of cancer cell proliferation and may have potential for use in colorectal cancer therapy.
Phosphatase and tensin homolog deleted on chromosome 10 (PTEN), a tumor suppressor gene, is located on chromosome 10q23.3 and is mutated at a high frequency in a variety of malignancies, including glioblastoma, melanoma, breast, prostate, and lung cancer (Li and Sun, 1997; Stambolic et al., 1998). The disruption of this gene in knockout mice results in the development of tumors (Di Cristofano et al., 1998). PTEN is a dual-specificity phosphatase and is able to act on both lipids and proteins. PTEN dephosphorylates phosphatidylinositol 3,4,5-triphosphate (PIP-3) (Di Cristofano et al., 1998), which is produced by phosphatidylinositol 3‐kinase (PI-3K), an important mediator of cell survival and proliferation (Myers et al., 1997; Stambolic et al., 1998). The overexpression of PTEN has been shown to suppress cell growth and tumor formation in nude mice. In addition, the disruption and mutation of PTEN in tumor cells results in the activation of protein kinase B (PKB)/AKT, which is activated via the PI-3K pathway cascade.
The p53 tumor suppressor protein is activated in response to cell stress and functions as a transcription factor to up-regulate gene products that are required for cell cycle arrest or apoptosis. For example, a transcription factor p53 induces G1 cell cycle arrest through transcriptional activation of p21WAF1 gene. p21WAF1 induced by p53 is associated with cyclin/CDK complexes to inhibit cell cycle progression (Harper et al., 1993; Xiong et al., 1993). Furthermore, the p21WAF1 protein has previously been shown to interact with the proliferating cell nuclear antigen (PCNA), thereby preventing DNA replication (Chuang et al., 1997). Recently published evidences indicate the existence of a mechanistic link between PTEN and p53 function through the control of the phosphorylation state of MDM2, which modulates the nuclear localization of MDM2 and the ubiquitination of p53 by the activation of PI-3K/AKT signal transduction pathway (Mayo and Donner, 2001; Mayo et al., 2002). MDM2, a RING finger ubiquitin ligase, is known to negatively regulate p53 via its capacity to bind to and mediate proteosomal degradation (Oliner et al., 1992, 1993; Haupt et al., 1997; Kubbutat et al., 1997).
PTEN-induced G1 cell cycle arrest in glioblastoma cells is associated with increased protein levels of the p27kip1, an inhibitor of the cyclin/CDK2 complex (Gottschalk et al., 2001). PTEN also resulted in the inhibition of cell cycle progression through negatively regulating PI-3K/AKT signaling pathway, and a target of this signaling process is the CDK inhibitor p27kip1 (Li and Sun, 1998). Furthermore, in PTEN (–/–) embryonic stem cells and fibroblast, inactivation of PTEN resulted in elevated levels of PIP-3, a product of PI-3K and accelerated G1/S transition that was accompanied by down-regulation of p27kip1 (Sun et al., 1999).
Gangliosides are sialic acid (NeuAc)-containing glycosphingolipids that are constituents of the plasma membranes of all vertebrate cells and are particularly abundant in the central nervous system (Svennerholm, 1980). Among the gangliosides, the ganglioside GM3 (Neu5Acα2,3 Galβ1,4Glcβ1,1Cer), one of the main components of the total gangliosides in many cell types, is synthesized in the first step of ganglioside biosynthesis, and all other ganglio-series gangliosides are synthesized from GM3 by linkage-specific glycosyltransferase (Van Echten and Sandhoff, 1993). In vitro, exogenously treated gangliosides are rapidly incorporated into the plasma membrane and are responsible for numerous biological effects (Laine and Hakomori, 1973). The simple ganglioside GM3 plays an important role in a variety of biological processes, such as cell–cell interactions, cell proliferation, and cell differentiation (Hakomori, 1990; Chung et al., 2005). Additionally, the epidermal growth factor (EGF)-mediated proliferation of epidermoid carcinoma cells is inhibited by ganglioside GM3 (Bremer et al., 1986). Ganglioside GM3 also suppresses the platelet-derived growth factor (PDGF)- and basic fibroblast growth factor (bFGF)-mediated proliferation of fibroblast cells and neuroblastoma cells (Yates et al., 1993; Hynds et al., 1995) and reduces proliferation and malignant potential of bladder cancer (Watanabe et al., 2002). Therefore, previous studies show that ganglioside GM3 is potentially associated with the inhibition of cancer cell proliferation. However, the precise function of ganglioside GM3 in anti-proliferation of cancer cells remains unclear.
In this study, we investigate whether ganglioside GM3 has an effect on the expression of tumor suppressor protein PTEN in colon cancer cells and whether ganglioside GM3-induced PTEN expression results in the inhibition of G1 cell cycle progression through up-regulation of p21WAF1 and p27kip1, CDK-negative regulators. We demonstrate that ganglioside GM3 results in the suppression of colon cancer cell proliferation by up-regulating PTEN expression and modulating expression of cell cycle regulatory protein.
Cellular localization of exogenously treated ganglioside GM3
To identify the ability to introduce exogenous ganglioside GM3 into the plasma membrane, immunohistochemical studies were carried out. No obvious immunofluorescence activity was observed in negative control experiments when a fluorescein isothiocyanate (FITC)-conjugated secondary antibody was used alone without the M2590 antibody in GM3-treated cells as well as in cells that had not been treated with GM3 (Figure 1A and B). However, immunofluorescence activity in GM3-treated cells became highly detected against GM3-untreated cells using M2590 and secondary antibodies (Figure 1C and D). It has been reported that gangliosides are located on the plasma membrane of mammalian cells (Laine and Hakomori, 1973; Svennerholm, 1980). Therefore, this result shows that exogenous ganglioside GM3 was successfully added to the plasma membrane.
Anti-proliferative effect and enhanced expression of PTEN by ganglioside GM3 in HCT116 colon cancer cells
Several groups have shown that ganglioside GM3 inhibits the proliferation of tumor cell lines (Bremer et al., 1986; Yates et al., 1993; Hynds et al., 1995; Watanabe et al., 2002). Thus, we examined whether ganglioside GM3 results in the decrease of HCT116 colon cancer cell proliferation. As shown in Figure 2A, ganglioside GM3 inhibited the growth of colon cancer cells in a dose-dependent manner. Additionally, the proliferation of HCT116 cells was decreased by 30 and 50 µM (data not shown) of ganglioside GM3 in a time-dependent manner.
In cancer models, the block in cell progression is closely associated with the accumulation of tumor suppressor PTEN (Myers et al., 1998; Stambolic et al., 1998). Previous results showed that ganglioside GM3 blocked HCT116 colon cancer cell proliferation (Figure 2A). On the basis of these results, we hypothesized that the inhibition of cell proliferation is associated with PTEN expression induced by ganglioside GM3 in colon cancer cells. Ganglioside GM3 resulted in the increase of PTEN mRNA expression in a dose-dependent manner, when HCT116 cells were exposed to various concentrations (0, 10, 20, 30 µM) of ganglioside GM3 (data not shown). The induction of PTEN gene mRNA increased dramatically for periods of up to 24 h at a concentration of 30 µM, as evidenced by reverse transcription–polymerase chain reaction (RT–PCR) and northern blot analysis. However, both ganglioside GM1 and lactosylceramide had no effect on the expression of PTEN mRNA and protein as determined by western and northern blot analyses (Figure 2B). Several studies have shown that transcriptional activation of PTEN gene is regulated by a transcription factor p53 protein (Stambolic et al., 1998; Chung et al., 2003). Thus, we further investigated whether ganglioside GM3 induces p53-dependent transcription of PTEN gene in HCT116 cells lacking p53. However, as shown in Figure 2B, ganglioside GM3 up-regulated PTEN expression in HCT116 p53(–/–) cells in a time-dependent manner, as seen in HCT116 cells. These results suggest that proliferation of HCT116 colon cancer cells was modulated by ganglioside GM3-stimulated PTEN expression through p53-independent mechanism.
The p53-dependent accumulation of p21WAF1 protein by ganglioside GM3-induced PTEN expression through the inhibition of PI-3K/AKT/MDM2 signaling pathway
The negative regulation of PKB/AKT-dependent cell survival by the tumor-suppressor PTEN has been reported previously (Myers et al., 1997; Di Cristofano et al., 1998; Stambolic et al., 1998). Mayo et al. (2002) have reported that the activation of PI-3K/AKT induces translocation of MDM2 from cytoplasm to nucleus where it negatively regulates p53. Additionally, PTEN restricts MDM2 to the cytoplasm and promotes MDM2 degradation and p53 function (Mayo and Donner, 2001). Our previous data showed that ganglioside GM3 up-regulated PTEN expression. Thus, we investigated whether ganglioside GM3 blocks the activation of PI-3K/AKT signaling on MDM2 stabilization and promotes p53 function in HCT116 colon cancer cells, using western blot analysis. As shown in Figure 3A, enhanced expression of PTEN in ganglioside GM3-treated HCT116 cells significantly induced the degradation of MDM2 protein by suppressing the activation of PI-3K/AKT signal pathway. Treatment of ganglioside GM3 in HCT116 cells also resulted in the induction of p53 stability through PTEN/PI-3K/AKT/MDM2 pathway. Furthermore, a transcription factor p53 induces G1 cell cycle arrest through transcriptional activation of p21WAF1 gene (Harper et al., 1993; Xiong et al., 1993). Thus, we examined the effect of GM3 on the expression of p21WAF1. As expected, p21WAF1 expression was clearly induced in ganglioside GM3-treated HCT116 cells. Moreover, PI-3K inhibitor wortmannin induced MDM2 degradation by blocking AKT activation without regulation of PTEN expression and also increased p21WAF1 expression by promoting p53 stability (Figure 3B). These results suggest that the induction of PTEN expression in ganglioside GM3-treated cells or PI-3K inhibitor-treated cells increases p21WAF1 induction for cell cycle arrest as well as the stability of p53 protein by facilitating MDM2 degradation through the inactivation of PI-3K/AKT pathway.
To better understand the roles of p53 in PTEN expression induced by ganglioside GM3, we took advantage of the availability of a panel of colon carcinoma isogenic cell lines, which lack p53(–/–). However, our preliminary data showed that ganglioside GM3-stimulated p53-independent PTEN expression as evidenced by RT–PCR and northern blot analysis (Figure 2B). As shown in Figure 4A, ganglioside GM3 in HCT116 p53(–/–) cells also resulted in the increase of PTEN expression, as evidenced by western blot analysis. Furthermore, previous data showed that PTEN expression stimulated by ganglioside GM3 in HCT116 cells stabilized p53, which induces transcriptional activation of p21WAF1 gene by blocking the activation of PI-3K/AKT/MDM2 pathway (Figure 3A). On the contrary, to further examine whether p53 levels, after ganglioside GM3 treatment, result in the increase of p21WAF1 expression, we checked p21WAF1 expression using ganglioside GM3-treated HCT116 p53(–/–) cells (Figure 4A). However, the expression of p21WAF1 was not increased in ganglioside GM3-treated HCT116 p53(–/–) cells, strongly suggesting that the induction of p21WAF1 is dependent on p53. To further confirm whether the ganglioside GM3-enhanced PTEN expression induces the transcriptional activation of the p21WAF1 using the p21WAF1 promoter containing the p53-binding site, we transiently transfected HCT116 cells with p21WAF1 promoter-driven luciferase reporter plasmids. As shown in Figure 4B, ganglioside GM3 significantly enhanced the transcriptional activity of the p21 full-length 2.4-kb promoter (p21P) by ∼7.5-fold, compared with the activity of the control vector (pGL3-basic). Moreover, we transfected HCT116 cell lines with the p21PΔ2.3 construct, a p21WAF1 construct lacking 250 bases from the 5′ end, corresponding to the p53 consensus DNA-binding site. Ganglioside GM3 markedly diminished the promoter activity of this construct (∼2.5-fold), compared with the activity of the p21P construct, indicating that the presence of functional p53 protein is required for the activation of the p21WAF1 promoter.
The induced expression of p27kip1 by ganglioside GM3
Several studies show that the PTEN tumor suppressor modulates G1 cell cycle progression through negatively regulating the PI-3K/AKT signaling pathway, and one critical target of this signaling process is the cyclin-dependent kinase (CDK) inhibitor (CKI) p27KIP1 (Li and Sun, 1998; Sun et al., 1999; Gottschalk et al., 2001). Thus, we investigate whether ganglioside GM3 up-regulates expression of p27KIP1 through the inhibition of PI-3K/AKT activation by regulating PTEN expression. As shown in Figure 5A, at 24 h after incubation of ganglioside GM3 in HCT116 cells, p27KIP1 expression was increased. Additionally, PI-3K inhibitor resulted in the induction of p27KIP1 expression by regulating AKT activation (Figure 5B). On the contrary, to investigate whether the expression of p27KIP1 by ganglioside GM3 is depended on the levels of p53 protein, we used HCT116-p53(–/–) cells. However, as shown in Figure 5A, ganglioside GM3 induced the expression of p27KIP1 in HCT116 cells lacking p53 gene. These results show that ganglioside GM3 results in the increase of p27KIP1 expression through p53-independent mechanism by inactivating PI-3K/AKT pathway, suggesting that ganglioside GM3 modulates PTEN expression.
p16INK4a binds and induces an allosteric conformational change in CDK4/CDK6 that inhibits the binding of adenosine triphosphate (ATP) and substantially reduces the formation of the CDK4/6-cyclin-D interface, which leads to the disruption of the interaction with D-type cyclins. This antagonizes cyclin binding and activation of CDK and induces G1 cell cycle arrest (Sherr and Roberts, 1999). Thus, we examined whether ganglioside GM3 also induces G1 cell cycle arrest by modulating p16INK4a. However, in both HCT116 cells and HCT116 lacking p53 cells, the levels of p16INK4a protein did not change by ganglioside GM3, compared with ganglioside GM3-untreated cells.
The inhibitory effect of ganglioside GM3 on the levels of cell cycle regulatory proteins for G1 cell cycle progression
The association of cyclin-CDK leads to activation and subsequent progression of the cell through the various phases of the cell cycle. Additionally, progression through the G1 phase requires association of D-type cyclins (Dl, D2, and D3) with CDK4 and CDK6 (Sherr, 1995), whereas cyclin E binding to CDK2 is required for the G1/S transition (Ohtsubo et al., 1995). Thus, we investigated whether ganglioside GM3 modulates expression of cell cycle regulatory proteins (CDK2, CDK4, cyclin D, and cyclin E) for G1 cell cycle arrest. As shown in Figure 6A, the protein levels of cyclin E and CDK2 were reduced by ganglioside GM3 in HCT116 cells, but not the protein levels of cyclin D1 and CDK4. Furthermore, to determine whether the down-regulation of cyclin E and CDK2 by ganglioside GM3 is due to the inhibition of PTEN-mediated PI-3K/AKT signaling, we treated the HCT116 colon cancer cells with PI-3K inhibitor. As shown in Figure 6B, wortmannin had no effect on the expression of cyclin E and CDK2 in HCT116 colon cancer cells. These results show that the suppression of cyclin E and CDK2 expression by ganglioside GM3 is not associated with PTEN-mediated PI-3K/AKT pathway.
Cell cycle arrest in G1 phase by ganglioside GM3
Because our previous data showed p53-dependent p21WAF1 expression by ganglioside GM3-induced PTEN expression for G1 cell cycle arrest, we further examined cell cycle distribution using a fluorescence-activated cell sorter (FACS) analysis in ganglioside GM3-treated cells. As shown in Figure 7, the HCT116 cells treated with ganglioside GM3 were arrested in the G1 phase of the cell cycle. The fraction of cells in the G1 phase increased from 62 to 90% after a 24 h treatment with ganglioside GM3. Furthermore, to determine that ganglioside GM3 do not promote G1 cell cycle arrest in HCT116 p53(–/–) cells, the DNA content of the HCT116 p53(–/–) cells treated with ganglioside GM3 was analyzed using flow cytometric analysis. However, G1 cell cycle arrest was induced in ganglioside GM3-treated HCT116 p53(–/–) cells, compared with ganglioside GM3-untreated HCT116 p53(–/–) cells, and G1 cell cycle arrest of ganglioside GM3-treated HCT116 p53(–/–) cells was somewhat reduced, compared with ganglioside GM3-treated HCT116 cells. These results suggest that ganglioside GM3 inhibits cell proliferation by arresting the cell cycle in the G1 phase because the expression of p27kip1, CDK2, and cyclin E as well as p53-dependent p21WAF1 was modulated by ganglioside GM3.
Effects of siRNA targeting PTEN gene on PTEN-mediated downstream regulation in ganglioside GM3-treated HCT116 cells
To confirm whether the suppression of PTEN levels by RNA interference restores the enhanced expression of p53-dependent p21WAF1 and p53-independent p27kip1 by suppressing the effect of PTEN on PI-3K/AKT pathway modulated by ganglioside GM3, small interfering RNA (siRNA) against PTEN and siRNA as negative control were utilized in ganglioside GM3-treated cells. As shown in Figure 8, the expression of PTEN induced by gangliosdie GM3 was repressed in cells transfected with siRNA targeting PTEN, compared with ganglioside GM3-exposed cells without PTEN siRNA or ganglioside GM3-treated cells transfected with negative control siRNA, as determined by western blot analysis. Furthermore, the suppression of PTEN expression by PTEN siRNA in ganglioside GM3-treated cells results in the increase of AKT phosphorylation compared with ganglioside GM3-treated cells without PTEN siRNA or ganglioside GM3-treated cells transfected with negative control siRNA. Moreover, PTEN siRNA restores the increased levels of p53, p21WAF1, and p27kip1 proteins through inhibiting the effect of PTEN on AKT pathway modulated by ganglioside GM3 in HCT116 colon cancer cells. These results clearly indicate that ganglioside GM3 regulates PTEN-mediated transcription of p53-dependent p21WAF1 and p53-independent p27kip1 for anti-proliferation of HCT116 colon cancer cells.
The lipid moiety of the exogenous gangliosides appears to be inserted into the lipid bilayer of the plasma membrane and not just absorbed to trypsin-sensitive membrane components, as has been proposed in other studies (Callies et al., 1977; Radsak et al., 1982; Schwarzmann et al., 1983). Because of this orientation, gangliosides have been implicated in a variety of cell-surface events such as recognition phenomena and the biotransduction of membrane-mediated information (Laine and Hakomori, 1973; Fishman and Brady, 1976; Moss et al., 1976; Hakomori, 1981; Fishman, 1982; Yamada et al., 1983; Hakomori, 1990). Our data show that ganglioside GM3 is localized on the surface of the membrane. It has recently been reported by numerous groups that the simple ganglioside GM3 acts as a growth regulator and inhibits proliferation and induces apoptosis (Laine and Hakomori, 1973; Hynds et al., 1995; Ono et al., 1999; Satoh et al., 2001; Watanabe et al., 2002; Wang et al., 2003; Chung et al., 2005). In addition, ganglioside GM3, which is localized in the membrane, is known to interact with other transmembrane proteins such as the motility-regulatory protein (CD9) and EGF receptor (EGFR) to form a complex, which facilitates cell adhesion, cell motility, and cell signaling (Ono et al., 1999, 2001; Wang et al., 2001). Although the inhibition of cell proliferation is induced by exogenously administered ganglioside GM3, the precise mechanism for this has not been fully explored.
The PTEN gene is frequently mutated in glioblastoma and in a significant fraction of prostate, endometrial, breast, lung, and other tumor types (Li et al., 1997; Maier et al., 1998). Inherited germline PTEN mutations are found in several rare autosomal dominant cancer predisposition syndromes, including Cowdens disease, Lhermitte–Duclos disease, and Bannayan–Zonana syndrome (Myers and Tonks, 1997; Cantley and Neel, 1999; Maehama and Dixon, 1999). It is well known that PTEN, a universal tumor suppressor gene, negatively regulates the PI-3K/AKT pathway during cell proliferation (Li and Sun, 1997; Myers et al., 1997; Di Cristofano et al., 1998; Stambolic et al., 1998; Sun et al., 1999). The activation of PI-3K/AKT in response to nerve growth factor, insulin-like growth factor-1, PDGF, interleukin-3, and the extracellular matrix promotes cell survival (Ahmed et al., 1997; Dudek et al., 1997; Songyang et al., 1997). Thus, AKT activation may be necessary and sufficient for cell survival, which emphasizes the significance of the negative regulatory effect of PTEN on this promiscuous survival factor. We have shown previously that the p53-mediated transcription of PTEN, a tumor suppressor, was inhibited in Hepatitis B Virus X gene-transfected liver cells, indicating the activation of AKT as a cell survival factor (Chung et al., 2003). Stambolic et al. (1998) show that p53 is an activator of PTEN transcription and that PTEN mRNA and protein levels increase in response to stimuli that result in p53 induction and that PTEN was required for p53-mediated apoptosis in immortalized mouse embryonic fibroblasts (Kubbutat et al., 1997). However, in this report, we demonstrate that ganglioside GM3, a growth regulator, results in the up-regulation of endogenous PTEN through p53-independent mechanism using p53-mutant HCT116 cells and suppresses the proliferation of HCT116 cells in time-dependent manner (Figure 2).
Tumor suppressor p53 controls the G1 and G2/M cell cycle checkpoints that mediate growth arrest (Agarwal et al., 1995). MDM2 binds to the N-terminal 42-amino acid transcriptional activation domain of p53 for its degradation (Fields and Jang, 1990) and negatively regulates the transcriptional functions of p53, including its capacity to induce cell cycle arrest. It has been shown that p53 induces the expression of p21WAF1, an inhibitor of CDKs, to control the cell cycle (Harper et al., 1993). The PTEN tumor suppressor inhibits PI-3K/AKT signaling that promotes translocation of MDM2 into the nucleus. When restricted to the cytoplasm, MDM2 is degraded. The ability of PTEN to inhibit the nuclear entry of MDM2 increases the cellular content and transactivation of the p53 tumor suppressor protein to promote the induction of p21WAF1 (Mayo and Donner, 2001). Thus, the findings herein show that the levels of p53 and p21WAF1 are increased in a time-dependent manner after ganglioside GM3 treatment and clearly show that the expression of PTEN induced by ganglioside GM3 is effective with respect to p53-dependent p21WAF1 accumulation for cell cycle arrest by inactivating PI-3K/AKT/MDM2 signaling in HCT116 cells (Figure 3). Because ganglioside GM3 significantly induced PTEN expression in both HCT116 and HCT116 p53(–/–) cells, as evidenced by RT–PCR and northern and western blot analyses (Figures 2–4), we assumed that ganglioside GM3-stimulated PTEN expression results in the induction of p21WAF1 expression and the increase of p53 protein through the inhibition of downstream function mediated by PI-3K/AKT pathway. Thus, to further determine whether ganglioside GM3-induced PTEN expression increases p53 protein-dependent p21WAF1 expression, we exposed a battery of isogenic cells, HCT116 p53(–/–), to ganglioside GM3 and clearly observed the up-regulation of PTEN without any changes in p21WAF1 expression in p53-deficient HCT116 cells. Therefore, our results have shown that ganglioside GM3 up-regulates expression of p21WAF1, which is dependent on the induction of p53 protein through the inactivation of PI-3K/AKT signaling by inducing PTEN expression. Moreover, we further investigated whether the increase in the stability of p53 is mediated by p21WAF1 transcription using p21WAF1 promoter which contains the 2.4 kb 5′-flanking region of the human p21WAF1 gene that includes the p53-binding site located at 2.3 kb and p53-binding-site-deleted p21WAF1 promoter. When HCT116 cells were transfected with the full-length promoter or with a construct lacking the p53-consensus sites, PTEN induced by ganglioside GM3 also transactivates the p21WAF1 promoter, and this transactivation requires p53. Although p53 is associated with PTEN expression as reported by several groups, our result provides clear evidence, suggesting that PTEN expression induced by ganglioside GM3 regulates the stability of p53 protein, and that p21WAF1 is dependent on p53 regulation. Because PTEN protects against p53 degradation by MDM2, a known ubiquitin ligase, p53 function is sustained in cells (Mayo et al., 2002). These results suggest that ganglioside GM3-induced PTEN expression requires p53-dependent p21WAF1 transcriptional activation to modulate cell cycle arrest.
On the basis of our previous findings, we further examined cell cycle distribution using an FACS analysis in ganglioside GM3-treated wild-type and p53(–/–) cells. As shown in Figure 7, we demonstrated that ganglioside GM3 results in the G1 cell cycle arrest in HCT116 cells compared with ganglioside GM3-untreated HCT116 cells. Because our previous data showed p53-dependent p21WAF1 expression by ganglioside GM3-induced PTEN expression for G1 cell cycle arrest, we assumed that ganglioside GM3 do not promote G1 cell cycle arrest in HCT116 p53(–/–) cells. However, although p53-dependent p21WAF1 expression was not increased in ganglioside GM3-treated HCT116 p53(–/–) cells (Figure 4), G1 cell cycle arrest was induced in ganglioside GM3-treated HCT116 p53(–/–) cells compared with ganglioside GM3-untreated HCT116 p53(–/–) cells, and cell cycle arrest of ganglioside GM3-treated HCT116 p53(–/–) cells was somewhat reduced when compared with ganglioside GM3-treated HCT116 cells. Thus, we guessed that the expression of PTEN induced by ganglioside GM3 had an effect on other factors for cell cycle arrest. It has recently been demonstrated that PTEN modulates G1 cell cycle progression and one critical target of this signaling process is the CKI p27kip1 by inactivating PI-3K/AKT signaling (Li and Sun, 1998; Sun et al., 1999; Gottschalk et al., 2001). As shown in Figure 5, our data show an elevated p27kip1 expression by ganglioside GM3-induced PTEN expression in HCT116 p53(–/–) cells as well as HCT116 cells through the suppression of PI-3K/AKT signal pathway. These results suggest that ganglioside GM3 modulates cell cycle progression by enhancing the expression of CDK inhibitors p21WAF1 and p27kip1 through the up-regulation of PTEN expression, showing the inactivation of cell survival PI-3K/AKT signaling in HCT116 colon cancer cells.
It is well known that G1-to-S cell cycle progression is controlled by several CDK complexes such as cyclin D1/CDK4 and cyclin E/CDK2; the activities of CDK complexes are dependent on the balance of cyclins and CKIs of p27kip1 and p21WAF1. To determine whether ganglioside GM3-induced G1 cell cycle arrest is due to the down-regulation of cyclins and CDKs or the up-regulation of CKIs in ganglioside GM3-treated cells, we analyzed the expression of these cell cycle regulators by a western blot analysis. The experiment indicated that ganglioside GM3-stimulated HCT116 cells resulted in the significant down-regulation of cyclin E/CDK2, but not cyclin D1/CDK4. Our data also show a significant up-regulation of p21WAF1 and p27kip1 during the G1-phase arrest through PTEN expression by ganglioside GM3. In addition, ganglioside GM3 led to an inhibition in the kinase activities associated with CDK2, using histone H1 substrate (data not shown).
In conclusion, as illustrated in Figure 9, we show that ganglioside GM3 induces a marked expression of PTEN, which blocks PI-3K/AKT survival signaling in colon cancer HCT116 cells. PTEN expression stimulated by ganglioside GM3 sustains the function of p53 as a transcriptional factor by inhibiting of MDM2 activity through the inactivation of PI-3K/AKT signal pathway. Ganglioside GM3-induced p53 protein also regulated the transcriptional activation of the p21WAF1 gene. Furthermore, ganglioside GM3-stimulated PTEN expression results in the induction of p27kip1 expression through PI-3K/AKT inactivation. Therefore, ganglioside GM3 inhibits proliferation of cancer cells by modulating the expression of cell cycle regulation factors. These results confirm the tumor-therapeutic potential of ganglioside GM3 and suggest that ganglioside GM3 may be an anti-tumor agent that would be useful in the treatment of highly proliferative human colorectal cancer.
Materials and methods
HCT116 colon carcinoma cells with wild-type p53 and their isogenic derivatives that lack p53 (HCT116 p53–/–) were provided by Dr. Young-Chae, Chang (School of Medicine, Catholic University of Daegu, Daegu, South Korea). HCT116 and HCT116 p53(–/–) cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS; JBI, Daegu, Korea), 100 unit/mL penicillin, and 100 µg/mL streptomycin, under an atmosphere of 5% CO2 at 37°C.
XTT proliferation assay
Cell proliferation was investigated using a commercially available proliferation kit II (XTT; Boehringer Mannheim, Mannheim, Germany). Briefly, the cells were subcultured in 96-well culture plates at a density of 103 cells/well in 100 µL of DMEM/10% FBS culture medium. After 24 h of incubation, the medium, in a 96-well plate, was discarded and replaced with 100 µL of new medium containing various concentrations of ganglioside GM3, or 30 µM ganglioside GM3. The plates were incubated in a 37°C humidified incubator under an atmosphere of 5% CO2 for 24 h, or various times (12, 24, and 48 h). At end of the incubation, 50 µL of XTT test solution prepared by mixing 5 mL of XTT-labeling reagent and 100 µL of electron-coupling reagent was added to each well. After 4 h of incubation at 37°C and under 5% CO2, the absorbance was measured on an ELISA reader (Molecular Devices, Sunnyvale, CA) at a test wavelength of 490 nm.
To confirm the distribution of exogenously treated ganglioside GM3, HCT116 colon cancer cells were seeded at a subconfluent density on sterile coverslips in six-well tissue culture plates. After incubating the attached cells in serum-free medium for 12 h, they were treated with 30 µM of ganglioside GM3 (Alexis, Sandiego, CA, USA) for 24 h. Ganglioside GM3-treated HCT116 cells were fixed in 3.7% formalin and washed three times with PBS. Non-specific sites were then blocked with 5% bovine serum albumin-containing PBS for 30 min at room temperature with gentle rocking. Thereafter, a solution of a ganglioside GM3-specific antibody (M2590; Biotest Laboratories, Tokyo, Japan) was flooded over the cells, and the cultures were incubated at 4°C overnight. After washing with PBS, the cells were further incubated with FITC-conjugated goat anti-mouse IgM (Biomeda, Foster City, CA) for 1 h at room temperature, followed by washing with PBS, and then analyzed using an Olympus BX50 fluorescence microscopy (Olympus, Tokyo, Japan). The pre-absorbed primary antibody or the secondary antibody alone was also carried out as a negative control experiment.
RT–PCR and northern blot analysis
Total RNA from each cell was isolated using the Trizol reagent (JBI), and the cDNAs were synthesized by reverse transcriptase with an oligo dT-adaptor prime from a Takara RNA PCR kit (Takara Shuzo, Shiba, Japan), according to the manufacturer’s recommended protocol. The cDNA was amplified by PCR with the following primers: PTEN (460 bp), 5′-TGCAATCCTCAGTTTGTGGT CTGCCA-3′ (sense) and 5′-GAAGTTGAACTGCTAGC CTCTGGATTTGA-3′ (antisense); β-actin (247 bp), 5′-CA AGAGATGGCCACGGCTGCT-3′ (sense) and 5′-TCCTT CTGCATCCTGTCGGCA-3′ (antisense). The use of equal amounts of mRNA in the RT–PCR assays was confirmed by analyzing the expression levels of β-actin. The PCR products were separated by gel electrophoresis on 2% agarose-containing ethidium bromide with 1× Tris-acetate-EDTA (TAE) buffer. A northern blot analysis was performed by the same method as described previously (Chung et al., 2003), using the [α-32P]dCTP-labeled fragments of the PTEN gene as a probe.
Western blot analysis
Cells were homogenized in a buffer containing 50 mM Tris–HCl (pH 8.0), 150 mM NaCl, 0.02% NaN3, 100 µg/mL phenylmethylsulfonyl fluoride (PMSF), 1 µg/mL aprotinin, and 1% Triton X-100. Protein concentrations were measured using the Bio-Rad protein assay (Bio-Rad, Richmond, CA). Twenty-microgram samples of total cell lysates were size fractionated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and electrophoretically transferred to nitrocellulose membranes using the Hoefer electrotransfer system (Amersham Biosciences, Buckinghamshire, UK). To detect target proteins, we incubated the membranes with the PTEN (NeoMarkers, Fremont, CA), p53 (Dako, Glostrup, Denmark), p21WAF1 (Becton Dickinson, Franklin Lakes, NJ), Cyclin D1 (Becton Dickinson), p27kip1 (Becton Dickinson), Mdm2 (Santa Cruz Biotechnology, Santa Cruz, CA), Cyclin E (Santa Cruz), CDK2 (Santa Cruz), CDK4 (Santa Cruz), p-AKT (Santa Cruz), p16INK4a (Calbiochem, San Diego, CA), and glyceraldehyde-3-phosphodehydrogenase (GAPDH) (Chemicon, Temecula, CA) antibodies, respectively. Detection was performed using a secondary horseradish peroxidase-linked anti-mouse antibody and an anti-rabbit antibody and the ECL chemiluminescence system (Amersham Biosciences).
Transfection and luciferase assay
The human p21WAF1 promoter constructs, p21P-luc (p21P) and p21PΔp53-luc reporter (p21PΔ2.3), have been described previously (Datto et al., 1995; Moon et al., 2003). For the reporter analysis of the p21WAF1 promoter, the transient transfection of HCT116 cells was carried out using the Lipofactamine reagent. Briefly, cells were plated on six-well plates at a density of 105 cells/well and allowed to grow overnight. The cells were co-transfected with 1 µg of p21WAF1 promoter-luciferase reporter constructs and 1 µg of β-galactosidase reporter plasmid by the LipofecAMINE method (Invitrogen). These cells were cultured in DMEM/10% FBS medium and incubated with ganglioside GM3 for 24 h. Luciferase and β-galactosidase activities were assayed using the luciferase and β-galactosidase enzyme assay system (Promega, Madison, WI). Luciferase activity was normalized with the β-galactosidase activity in cell lysates and calculated as an average of three independent experiments.
Analysis of DNA content by flow cytometry
Samples of HCT116 cells (2 × 106 cells) were centrifuged (2500 × g, 4°C, 10 min) and washed twice in PBS buffer. The pellet was gently resuspended in 100 µL of PBS, and 200 µL of PBS containing 10% ethanol/5% glycerol was then added, followed by 200 µL of PBS containing 50% ethanol/5% glycerol, and the samples were then incubated on ice for 5 min. A total of 1 mL of PBS containing 70% ethanol/5% glycerol was added to the cell suspension and left at 4°C overnight, washed with PBS, and resuspended with 12.5 µg of RNase (Sigma, St. Louis, MO) in 250 µL of 1.12% sodium citrate buffer (pH 8.45). Incubation was continued at 37°C for 30 min before staining the cellular DNA with 250 µL of propidium iodide (50 µg/mL) for 30 min at room temperature. The stained cells were analyzed on a FACScan flow cytometer for relative DNA content, on the basis of increased red fluorescence.
Samples of the total protein (100 µg) were incubated with the anti-CDK2 and anti-Cyclin E polyclonal antibodies for 2 h at 4°C, followed by incubation with 10 µL of the protein A/G–agarose beads (Santa Cruz) for 1 h. The protein complexes were washed four times with an immunoprecipitation buffer (50 mM Tris–HCl [pH 7.4], 0.5% NP-40, 150 mM NaCl, 50 mM NaF, 0.2 mM sodium orthovanadate, 1 mM dithiothreitol [DTT], 20 µg/mL aprotinin, 20 µg/mL leupeptin, 1 mM phenylmethylsulfonyl fluoride) and released from the beads by boiling in 2× SDS sample buffer (125 mM Tris–HCl [pH 6.8], 4% SDS, 10% β-mercaptoethanol, 2% glycerol, 0.02% bromophenol blue) for 5 min. The reaction mixture was then resolved by a 12% SDS–PAGE, transferred onto a nitrocellulose membrane by electroblotting, and probed with the anti-CDK2 and anti-Cyclin E antibodies. The blot was developed using an ECL kit.
Preparation and transfection of siRNAs
Each of siRNA duplex was designed to target the coding sequence of human PTEN mRNA and plant chlorophyll a/b-binding protein mRNA as a negative control and synthesized by Bioneer Corporation (Daejeon, South Korea). The target sequence of PTEN siRNA is 5′-GAUAUCAAGA GAUGGAUU-3′. HCT116 colon cancer cells were transfected with PTEN siRNA and negative control siRNA respectively by using Lipofectamine 2000 (Invitrogen), according to the manufacturer’s instructions. One day after transfection, transfection complexes were removed and replaced with culture medium. After incubating with ganglioside GM3 in culture medium for 24 h, the transfected cells were used for experiments.
Conflict of interest statement
These authors contributed equally to this work.
This work was supported by National Research Laboratory Program (M10203000024-02J0000-01300) from the Ministry of Science and Technology, Korea (C.H.K.).
2Molecular and Cellular Glycobiology Unit, Department of Biological Science, Sungkyunkwan University, 300 Chunchun-Dong, Jangan-Gu, Suwon City, Kyunggi-Do 440-746, South Korea; 3Department of Microbiology, Kyungpook National University, Daegu 702-701, South Korea; 4Faculty of Biotechnology, Dong-A University, Saha-Gu, Busan 604-714, South Korea; and 5Systemic Proteomics Research Center, Korea Research Institute of Bioscience and Biotechnology, Taejon 305-600, South Korea