The in vitro and in vivo effects of 2-(4-morpholinyl)-8-phenyl-chromone (LY294002), a specific inhibitor of phosphatidylinositol 3'-kinase, in human colon cancer cells.

PURPOSE: Phosphatidylinositol 3'-kinase (PI3K) and Akt/protein kinase B(PKB) allow for escape from apoptosis in various human cancer cells. We postulated that 2-(4-morpholinyl)-8-phenyl-chromone (LY294002), a PI3K inhibitor, should inactivate Akt/PKB, consequently inhibiting cell proliferation and inducing apoptosis in vitro and in vivo. EXPERIMENTAL DESIGN: Human colon cancer cell lines (DLD-1, LoVo, HCT15, and Colo205) and their mouse xenografts (DLD-1 and LoVo) were used in this study. The expression of phosphorylated Akt (Ser(473)) and apoptosis in cancer cells were determined by immunoblotting and immunohistochemistry. To evaluate the activity of caspase-3 in culturing cells, the caspase colorimetric assay was also performed. RESULTS: LY294002 demonstrated a remarkable growth-inhibitory and apoptosis-inducing effect in these colon cancer cell lines, with decreased expression of phosphorylated Akt (Ser(473)). However, there was a great discrepancy between the sensitivity for LY294002 and the level of expression of phosphorylated Akt. Although the LoVo and Colo205 cells exhibited high sensitivity to LY294002 with increased apoptosis, the DLD-1 and HCT15 cells did not show rapid induction of apoptosis. The caspase-3 activity was significantly high in the LoVo cells but not in the DLD-1 cells. In the experiments using mouse xenografts, we found that LY294002 administration in vivo also resulted in suppression of tumor growth and induction of apoptosis, especially in the LoVo tumors, and therefore showed remarkable effectiveness in the mouse peritonitis carcinomatosa model. CONCLUSIONS: PI3K-Akt/PKB plays an important role in colon cancer development and progression by helping to promote cell growth and allowing cells to escape apoptosis. These results propose the usefulness of LY294002 as an antitumoral agent for patients with colorectal cancer.     

In vitro and in vivo studies of the anticancer action of terbinafine in human cancer cell lines: G0/G1 p53-associated cell cycle arrest

Terbinafine (TB) (Lamisil), a promising oral antifungal agent used worldwide, has been used in the treatment of superficial mycosis. In our study, we demonstrated that TB dose-dependently decreased cell number in various cultured human malignant cells. Flow cytometry analysis revealed that TB interrupts the cell cycle at the G0/G1 transition. The TB-induced cell cycle arrest in colon cancer cell line (COLO 205) occurred when the cyclin-dependent kinase (cdk) system was inhibited just as the levels of p53, p21/Cip1 and p27/Kip1 proteins were augmented. In the TB-treated COLO 205, the binding between p53 protein and p53 consensus binding site in p21/Cip1 promoter DNA probe was increased. Pretreatment of COLO 205 with p53-specific antisense oligodeoxynucleotide decreased the TB-induced elevations of p53 and p21/Cip1 proteins, which in turn led to arrest in the cell cycle at the G0/G1 phase. Moreover, in the p53 null cells, HL60, TB treatment did not induce cell cycle arrest. Taken together, these results suggest an involvement of the p53-associated signaling pathway in the TB-induced antiproliferation in COLO 205. We further examined whether administration of TB could affect the growth of tumors derived from human colon cancer cells in an in vivo setting. COLO 205 cells implanted subcutaneously in nude mice formed solid tumor; subsequent intraperitoneal injections of TB (50 mg/kg) led to obvious decline in tumor size, up to 50-60%. In these tumors, increases in the p21/Cip1, p27/Kip1 and p53 proteins and the occurrence of apoptosis were observed. Combined treatment with TB and nocodazole (ND), a clinically used anticancer agent, potentiated the apoptotic effect in COLO 205. These findings demonstrate for the first time that TB can inhibit the proliferation of tumor cells in vitro and in vivo.     

Study of in vitro and in vivo effects of 1,6-Bis[4-(4-amino-3-hydroxyphenoxy)phenyl]diamantane (DPD), a novel cytostatic and differentiation inducing agent, on human colon cancer cells

A diamantane derivative 1,6-Bis [4-(4-amino-3-hydroxyphenoxy) phenyl] diamantane (DPD) was found to inhibit the growth of several cancer cell lines in the National Cancer Institute (NCI) Anticancer Drug Screen system. In this study, we examined the in vitro and in vivo effects of DPD on human colon cancer cells. DPD exerted growth inhibitory activities in vitro against three human colon cancer cell lines (Colo 205, HT-29, and HCT-15). DPD-treated cells were arrested at G(0)/G(1) as analysed by flow cytometric analysis. The expression of cyclin D was decreased in DPD-treated cells. The differentiation markers of carcinoembryonic antigen and fibronectin were significantly increased in colon cancer cells after treatment with DPD. The epithelium-like brush borders on HT-29 cell surface were also demonstrated at 1 week after withdrawal from DPD treatment. The DPD-induced cell growth inhibition and differentiation were irreversible after removal of DPD. The in vivo effect of tumour growth suppression by DPD was also observed in mouse xenografts. No acute toxicity was observed after an intraperitoneal challenge of DPD in BALB/c-nude mice weekly. These results suggest that DPD appears to be a new potentially less toxic modality of cancer therapy.     

Treatment of human colon cancer xenografts with TRA-8 anti-death receptor 5 antibody alone or in combination with CPT-11.

PURPOSE: This study was designed to evaluate the in vitro cytotoxicity and in vivo efficacy of TRA-8, a mouse monoclonal antibody that binds to the DR5 death receptor for tumor necrosis factor-related apoptosis-inducing ligand (also called Apo2L), alone and in combination with CPT-11, against human colon cancer cells and xenografts. EXPERIMENTAL DESIGN: DR5 expression was assessed on human colon cancer cell lines using flow cytometry, and cellular cytotoxicity after TRA-8 treatment, alone and in combination with SN-38, was determined by measuring cellular ATP levels. Tumor growth inhibition and regression rates of well-established subcutaneous COLO 205, SW948, HCT116, and HT-29 colon cancer xenografts in athymic nude mice treated with TRA-8 or CPT-11 alone and in combination were determined. (99m)Tc-TRA-8 was used to examine tumor localization of TRA-8 in animals bearing each of the four xenografts. In addition, whole-body biodistribution and imaging was carried out in COLO 205-bearing animals using in vivo single-photon emission computed tomography imaging and tissue counting. RESULTS: DR5 expression was highest on HCT116, intermediate on SW948 and COLO 205 cells, and lowest on HT-29 cells. COLO 205 cells were the most sensitive to TRA-8-induced cytotoxicity in vitro, SW948 and HCT116 cell lines were moderately sensitive, and HT-29 cells were resistant. Combination treatment with TRA-8 and SN-38 produced additive to synergistic cytotoxicity against all cell lines compared with either single agent. The levels of apoptosis in all cell lines, including HT-29, were increased by combination treatment with SN-38. In vivo, combination therapy with TRA-8 and CPT-11 was superior to either single-agent regimen for three of the xenografts: COLO 205, SW948, and HCT116. COLO 205 tumors were most responsive to therapy with 73% complete regressions after combination therapy. HT-29 cells derived no antitumor efficacy from TRA-8 therapy. Tumor xenografts established from the four colon cancer cell lines had comparable specific localization of (99m)Tc-TRA-8. CONCLUSIONS: In vitro and in vivo effects of TRA-8 anti-DR5 monoclonal antibody on four different colon cancer cell lines and xenografts were quite variable. The HT-29 cell line had low surface DR5 expression and was resistant to TRA-8 both in vitro and in vivo. Three cell lines (COLO 205, SW948, and HCT116) exhibited moderate to high sensitivity to TRA-8-mediated cytotoxicity which was further enhanced by the addition of SN-38, the active metabolite of CPT-11. In vivo, the combination of TRA-8 and CPT-11 treatment produced the highest antitumor efficacy against xenografts established from the three TRA-8-sensitive tumor cell lines. All four colon cancer xenografts had comparable localization of (99m)Tc-TRA-8. These studies support the strategy of TRA-8/CPT-11 combined treatment in human colon cancer clinical trials.     

Effect of Sodium Butyrate on p16INK4a,p14ARF,p15INK4b,Class I HDACs (HDACs 1, 2, 3) Class II HDACs (HDACs 4, 5, 6), Cell Growth Inhibition and Apoptosis Induction in Pancreatic Cancer AsPC-1 and Colon Cancer HCT-116 Cell Lines

Background: In higher eukaryotes, cell-cycle transitions are regulated by different cyclin-dependent kinases (Cdks) and Cdk inhibitors (CKIs). CKIs include two groups, the Ink4 (p16INK4a, p15INK4b, p18INK4c, and p19INK4d) and the Cip/Kip (p21Cip1, p27Kip1, and p57Kip2) families. The hyperactivity of histone deacetylases (HDACs) is associated with cancer induction. Histone deacetylase inhibitors (HDACIs) such as sodium butyrate (NaBT) can inhibit HDAC activity resulting in apoptosis induction. The present study was designed to investigate the effect of sodium butyrate on p16INK4a, p14ARF, p15INK4b, class I HDACs (HDACs 1, 2, 3), and class II HDACs (HDACs 4, 5, 6), cell growth inhibition, and apoptosis induction in pancreatic cancer AsPC-1 and colon cancer HCT-116 cell lines. In fact, we want to know whether sodium butyrate can reactivate Ink4 and Cip/Kip families by HDACs inhibition. Materials and Methods: The AsPC-1 and HCT-116 cells were treated with sodium butyrate at different periods. Then, the MTT assay, cell apoptosis assay, and qRT-PCR were done to determine viability, apoptosis, and the relative expression level of the genes respectively. Results: The sodium butyrate increased p16INK4a, p14ARF, and p15INK4b and decreased class I and II HDACs significantly. Besides, HCT-116 cell was more sensitive to sodium butyrate in comparison to AsPC-1 cell. Conclusion: The sodium butyrate can reactivate the p16INK4a, p14ARF, and p15INK4b through inhibition of HDACs in AsPC-1 and HCT-116 cell lines.     

Investigation of the Effect of Zebularine in Comparison to and in Combination with Trichostatin A on p21Cip1/Waf1/ Sdi1, p27Kip1, p57Kip2, DNA Methyltransferases and Histone Deacetylases in Colon Cancer LS 180 Cell Line

Background: The heart of the cell cycle regulatory machine is a group of enzymes named cyclin-dependent kinases (Cdks). The active form of these enzymes includes a kinase and its partner, a cyclin. The regulation of cyclin-Cdk complexes is provided by Cdk inhibitors (CKIs) such as Cip/Kip family comprising p21Cip1/Waf1/Sdi1, p27Kip1, and p57Kip2. The hypermethylation and deacetylation of Cip/Kip gene family seem to be frequent in numerous cancers. It has been indicated that increased expression of DNMTs and HDACs contributes to cancer induction. Previously, we reported the effect of DNA demethylating agents and histone deacetylase inhibitors on histone deacetylase 1, DNA methyltransferase 1, and CIP/KIP family in colon cancer. The current study was designed to evaluate the effect of zebularine in comparison to and in combination with trichostatin A (TSA) on p21Cip1/Waf1/Sdi1, p27Kip1, p57Kip2, DNA methyltransferases (DNMT1, 3a and 3b) and histone deacetylases (HDAC1, 2, and 3) genes expression, cell growth inhibition and apoptosis induction in colon cancer LS 180 cell line. Materials and Methods: The colon cancer LS 180 cell line was cultured and treated with zebularine and TSA. To determine cell viability, apoptosis, and the relative expression level of the genes, MTT assay, cell apoptosis assay, and qRT-PCR were done respectively. Results: Both compounds significantly inhibited cell growth, and induced apoptosis. Furthermore, both compounds increased p21Cip1/Waf1/Sdi1, p27Kip1, and p57Kip2 significantly. Additionally, zebularine and TSA decreased DNMTs and HDACs gene expression respectively. Conclusion: The zebularine and TSA can reactivate the CIP/KIP family through inhibition of DNMTs and HDACs genes activity.     

Effects of alpha-difluoromethylornithine and 5-fluorouracil on the proliferation of a human colon adenocarcinoma cell line

Because alpha-difluoromethylornithine (DFMO) reduces the incidence of experimental colon cancers, inhibits the growth of human lung cancer cells and human leukemia cells in culture, and in combination with methylglyoxal (bis)guanylhydrazone induces remission in children with leukemia, its effectiveness against a human colon adenocarcinoma cell line (Colo 205) was tested alone and in combination with 5-fluorouracil (5-FU). Both DFMO (2 X 10(-4) M) and 5-FU (10(-6) M) inhibited Colo 205 cell proliferation. Above 5 X 10(-4) M DFMO (p less than 0.001) and at 10(-4) M 5-FU (p less than 0.001), Colo 205 growth was completely inhibited. Although DFMO did not sensitize Colo 205 cells to a noninhibitory concentration of 5-FU, the effectiveness of inhibitory concentrations of 5-FU and DFMO in reducing Colo 205 cell growth was additive. DFMO (2 X 10(-4) M) caused 89 to 93% inhibition of ornithine decarboxylase activity (p less than 0.001) and reduced levels of putrescine (93%; p less than 0.01) and spermidine (57%; p less than 0.02). Growth rate and the intracellular putrescine and spermidine contents were restored by 10(-6) M putrescine. DFMO could be an effective chemotherapeutic agent against human colonic cancer because of its effects at such unusually low concentrations in vitro.     

Detection of poly(ADP-ribose) polymerase cleavage in response to treatment with topoisomerase I inhibitors: a potential surrogate end point to assess treatment effectiveness.

Cleavage of poly(ADP-ribose) polymerase (PARP) by caspases is a prominent characteristic of apoptosis or programmed cell death shown to be induced by topoisomerase (Topo) inhibitors. Because Topo I inhibitors have been shown to be effective in the treatment of some patients with colon cancer, we considered the possibility of using PARP cleavage as an early predictor of responsiveness to this class of agents. We show cleavage of PARP in response to treatment with Topo I inhibitors in colon cancer both in vitro and in vivo: (a) in vitro in SW480, HCT116, VACO5, VACO6, VACO8, VACO411, VACO425, and VACO451 human colon cancer cell lines treated with topotecan (TPT) or CPT-11; (b) in vivo in SW480, VACO451, and VRC5 colon cancer xenografts grown in athymic mice treated with TPT or CPT-11; and (c) in vivo in colon cancer samples from patients undergoing a Phase II clinical trial with CPT-11. Our results show a strong correlation between percentage of PARP cleavage and percentage of acridine orange-positive cells in colon cancer cell lines treated with 0.1 microM TPT for 24 and 48 h, confirming that PARP cleavage is a useful marker for programmed cell death in colon cancer cell lines. Results from experiments performed on colon cancer xenografts also show an association between PARP cleavage and response to treatment with TPT or CPT-11. The increase of PARP cleavage in xenografts and in clinical samples corresponding to treatment with Topo I inhibitors suggests that this procedure may have early predictive value to assess effectiveness of treatment. These results provide the basis for determining the validity of using PARP cleavage as an early marker of chemotherapeutic effectiveness in human samples.     

Effect of Zebularine in Comparison to and in Combination with Trichostatin A on CIP/KIP Family (p21Cip1/Waf1/Sdi1, p27Kip1, and p57Kip2), DNMTs (DNMT1, DNMT3a,and DNMT3b), Class I HDACs (HDACs 1, 2, 3) and Class II HDACs (HDACs 4, 5, 6) Gene Expression,Cell Growth Inhibition and Apoptosis Induction in Colon Cancer LS 174T Cell Line

Background: A pattern of epigenetic modifications and changes, DNA methylation and histone modification, is central to many human cancers. A variety of tumor suppressor genes (TSGs) have been demonstrated to be silenced because of histone deacetylation and DNA hypermethylation in several cancers. Recent in vitro studies have shown that two known mechanisms of epigenetic alteration consisting of methylation and histone deacetylation seem to be the best candidate mechanisms for inactivation of CIP/KIP family (p21Cip1/Waf1/Sdi1, and p27Kip1) in numerous cancers. Numerous investigations have indicated that DNA demethylating and histone deacetylase inhibitors (HDACIs) can restore the CIP/KIP family gene expression. Previously, we evaluated the effect of trichostatin A (TSA) and 5-aza-2′-deoxycytidine (5-AZA-CdR) on hepatocellular carcinoma (HCC). The present study was designed to investigate the effect of zebularine in comparison to and in combination with trichostatin A on p21Cip1/Waf1/Sdi1, p27Kip1, p57Kip2, DNMT1, DNMT3a and DNMT3b, Class I HDACs (HDACs 1, 2, 3) and Class II HDACs (HDACs 4, 5, 6) gene expression, cell growth inhibition and apoptosis induction in colon cancer LS 174T cell line. Materials and Methods: The colon cancer LS 174T cell line was cultured and treated with zebularine and TSA. To determine cell viability, apoptosis, and the relative expression level of the genes, MTT assay, cell apoptosis assay, and qRT-PCR were done respectively. Results: Both compounds significantly inhibited cell growth, and induced apoptosis. Furthermore, both compounds increased p21Cip1/Waf1/Sdi1, p27Kip1, and p57Kip2 significantly. Additionally, zebularine and TSA decreased DNMTs and HDACs gene expression respectively. Conclusion: The zebularine and trichostatin A can reactivate the CIP/KIP family through inhibition of DNMTs and HDACs genes activity.     

Artepillin C in Brazilian propolis induces G(0)/G(1) arrest via stimulation of Cip1/p21 expression in human colon cancer cells

Potential chemopreventive agents exist in foods. Artepillin C in Brazilian propolis was investigated for its effects on colon carcinogenesis. We had found that artepillin C was a bioavailable antioxidant, which could be incorporated into intestinal Caco-2 and hepatic HepG2 cells without any conjugation and inhibited the oxidation of intracellular DNA. Artepillin C was then added to human colon cancer WiDr cells. It dose-dependently inhibited cell growth, inducing G(0)/G(1) arrest. The events involved a decrease in the kinase activity of a complex of cyclin D/cyclin-dependent kinase 4 and in the levels of retinoblastoma protein phosphorylated at Ser 780 and 807/811. The inhibitors of the complex, Cip1/p21 and Kip1/p27, increased at the protein level. On the other hand, Northern blotting showed that artepillin C did not affect the expression of Kip1/p27 mRNA. According to the experiments using isogenic human colorectal carcinoma cell lines, artepillin C failed to induce G(0)/G(1) arrest in the Cip1/p21-deleted HCT116 cells, but not in the wild-type HCT116 cells. Artepillin C appears to prevent colon cancer through the induction of cell-cycle arrest by stimulating the expression of Cip1/p21 and to be a useful chemopreventing factor in colon carcinogenesis.     

Cdk2 is dispensable for cell cycle inhibition and tumor suppression mediated by p27(Kip1) and p21(Cip1)

p27(Kip1) and p21(Cip1) are thought to suppress tumor growth and prevent cell cycle progression by inhibiting Cdk2-cyclin E/A kinases. Since Cdk2 is dispensable for mitotic cell division, we analyzed the activity of these inhibitors in Cdk2-deficient cells. Ectopic expression of p27(Kip1) or p21(Cip1) efficiently inhibits cell cycle progression of Cdk2(-/-) fibroblasts. Loss of p27(Kip1) or p21(Cip1) confers similar proliferative advantages to Cdk2(+/+) and Cdk2(-/-) cells. Moreover, Cdk2 is dispensable for p21(Cip1)-induced cell cycle arrest after DNA damage. Finally, ablation of Cdk2 in p27(Kip1) null mice does not suppress their phenotypic defects, including development of pituitary tumors. These results indicate that Cdk2 is not an essential target for p27(Kip1) and p21(Cip1) in cell cycle inhibition and tumor suppression.     

19-nor-1 alpha,25-dihydroxyvitamin D2 (paricalcitol) inhibits the proliferation of human pancreatic cancer cells in vitro and in vivo

1,25-dihydroxyvitamin D(3), (1,25(OH)(2)D(3); calcitriol), the hormonal form of vitamin D, exerts growth-inhibitory, pro-apoptotic and anti-metastatic effects on tumor cells in vitro and in vivo but its clinical use is limited by its calcemic effects. Previous studies have shown that the antiproliferative effects of the less calcemic calcitriol analog 19-nor-1,25-(OH)(2)D(2) (paricalcitol) on prostate tumor cell lines are indistinguishable from those of 1,25(OH)(2)D(3). We therefore investigated the anti-proliferative effects of paricalcitol on the growth of pancreatic tumor cell lines in vitro and in vivo. Both 1,25(OH)(2)D(3) and paricalcitol inhibited the growth of BxPC-3, Hs700T and AsPC-1 lines in a dose-dependent manner. This antiproliferative activity correlated with upregulation of the cell cycle inhibitors p21 (Waf1/CIP1) and p27(Kip1). A fourth pancreatic cell line, Hs766T was unresponsive to both paricalcitol and calcitriol. Hs766T cells also failed to upregulate p21/Waf-1/Cip1 or p27/KiP in response to treatments with these agents. Paricalcitol, given three times per week inhibited the growth of AsPC-1 pancreatic tumor cell xenografts in nude mice at a dose that did not cause hypercalcaemia. Tumor inhibition was accompanied by in vivo upregulation of p21 and p27 expression. Given the few therapeutic options for patients with pancreatic cancer, further exploration of paricalcitol, an FDA-approved medication, is warranted.     

C1B domain peptide of protein kinase Cγ significantly suppresses growth of human colon cancer cells in vitro and in an in vivo mouse xenograft model through induction of cell cycle arrest and apoptosis

Two peptides derived from the C1B domain of protein kinase Cγ (PKCγ) were shown to associate with classical PKC isozymes and modulate their activities. These C1B peptides are designated C1B1 (amino acid residues 101-112) and C1B5 (residues 141-151). Since PKC enzyme activity is shown to be involved in colon cancer development, the effect of C1B peptides on the growth of various human colon cancer cell lines was examined in vitro and in vivo. Sub-micromolar to micromolar levels of both C1B peptides induced approximately 60-70% growth attenuation in multiple colon cancer cell lines in a soft agar tumor colony assay; however, C1B5 peptide was not cytotoxic to normal colon epithelial cells in two dimensional culture. The effect of C1B5 peptide on colony growth of COLO205 cells was reversed by treatment with the PKCα/β inhibitor, Ro-32-0432. C1B peptide treatment attenuated COLO205 cells via two mechanisms: 1) cell cycle arrest and 2) stimulation of apoptosis. This is evident in G 2 arrest and increases in levels of cleaved caspase 3 and p53 phosphorylated at serine 20. Intratumoral injection of C1B5 peptide (20 mg/kg/day, every three days) markedly attenuated the growth of subcutaneous xenografts of COLO205 cells in SCID mice by 76% compared with the control. Taken together, these results strongly suggest that C1B peptides have negligible effects on normal tissues but are potentially effective chemotherapeutic agents for colon cancer.     

Involvement of both extracellular signal-regulated kinase and c-jun N-terminal kinase pathways in the 12-O-tetradecanoylphorbol-13-acetate-induced upregulation of p21(Cip1) in colon cancer cells

Protein kinase C (PKC), a family of serine-threonine kinases, has been implicated in the regulation of colon tumorigenesis. However, the specific isoform of PKC involved in this process is not clear. In the present study, we found that treatment of the cultured human colon cancer cell line COLO-205 with a PKC agonist, 12-O-tetradecanoylphorbol-13-acetate (TPA), resulted in cell-cycle arrest at the G(0)/G(1) phase, decrease in cell number, PKCgamma isoform translocation, and upregulation of p21(Cip1) protein. Pretreatment of the cells with a PKC inhibitor, staurosporine, prevented the TPA-induced upregulation of p21(Cip1) protein. Based on the findings of the present study including that (a) both extracellular signal-regulated kinase (ERK) and c-jun N-terminal kinase (JNK) were activated in the TPA-treated COLO-205 cells, (b) pretreatment with the mitogen-activated protein kinase kinase inhibitor PD98059 but not with the p38 mitogen-activated protein kinase inhibitor SB203580 blocked the TPA-induced p21(Cip1) in COLO-205 cells, and (c) transient transfection of the COLO-205 cells with dominant negative ERK or JNK plasmid significantly suppressed the TPA-induced p21(Cip1) protein induction, we conclude that both the ERK and JNK pathways are involved in the TPA-induced upregulation of p21(Cip1) protein in the COLO-205 cells.     

Molecular mechanisms of G0/G1 cell-cycle arrest and apoptosis induced by terfenadine in human cancer cells

Terfenadine (TF), a highly potent histamine H1 receptor antagonist, has been shown to exert no significant central nervous system side effects in clinically effective doses. In this study, we demonstrated that TF induced significant growth inhibition of human cancer cells, including Hep G2, HT 29, and COLO 205 cells, through induction of G(0)/G(1) phase cell-cycle arrest. The minimal dose of TF induced significant G(0)/G(1) arrest in these cells was 1-3 microM. The protein levels of p53, p21/Cip1, and p27/Kip1 were significantly elevated, whereas the kinase activities of cyclin-dependent kinase 2 (CDK2) and CDK4 were inhibited simultaneously in the TF-treated cells. On the other hand, significant apoptosis, but not G(0)/G(1) arrest, was induced in the HL 60 (p53-null) or Hep 3B (with deleted p53) cells when treated with TF (3-5 microM). To clarify the roles of p21/Cip1 and p27/Kip1 protein expression, which was involved in G(0)/G(1) arrest and apoptosis induced by TF in human cancer cells, antisense oligodeoxynucleotides (ODNs) specific to p21/Cip1 and p27/Kip1 were used, and the expression of the p21/Cip1 and p27/Kip1 were monitored by immunoblotting analysis. Our data demonstrated that the percentage of the apoptotic cells detected by annexin V/PI analysis in the TF-treated group was clearly attenuated by pretreatment with p27/Kip1-specific ODNs. These results indicated that p27/Kip1 (but not p21/Cip1) protein indeed played a critical role in the TF-induced apoptosis. We also demonstrated that the TF-induced G(0)/G(1) cell-cycle arrest effect was not reversed by TF removal, and this growth inhibition lasted for at least 7 d. Importantly, the occurrence of apoptosis and cell growth arrest was not observed in the TF-treated normal human fibroblast, even at a dose as high as 25 microM. Our study showed the molecular mechanisms for TF-induced cell growth inhibition and the occurrence of apoptosis in human cancer cells.     

Anticancer effects of low-dose 10-hydroxycamptothecin in human colon cancer

10-Hydroxycamptothecin (10-HCPT), an indole alkaloid isolated from a Chinese tree, Camptotheca acuminate, inhibits the activity of topoisomerase I and has a broad spectrum of anticancer activity in vitro and in vivo. However, its use has been limited due to its water-insolubility and toxicity with i.v. administration. The purpose of this study was to investigate the efficacy, toxicity and proper dosage of 10-HCPT as a single agent by oral administration in the treatment of human colon cancer. 10-HCPT significantly repressed the proliferation of Colo 205 cells at a relatively low concentration (5-20 nM). Flow cytometry analysis and western blot and apoptosis assays demonstrated that low-dose 10-HCPT arrested Colo 205 cells in the G2 phase of the cell cycle and triggered apoptosis through a caspase-3-dependent pathway. Moreover, following oral administration at doses of 2.5-7.5 mg/kg/2 days, significant suppression of tumor growth by 10-HCPT was observed in mouse xenografts. No acute toxicity was observed after an oral challenge of 10-HCPT in BALB/c-nude mice every 2 days. The results of this study suggest that a relatively low dose of 10-HCPT (p.o.) is able to inhibit the growth of colon cancer, facilitating the development of a new protocol of human trials with this anticancer drug.     

Acute tumour response to the MEK1/2 inhibitor selumetinib (AZD6244, ARRY-142886) evaluated by non-invasive diffusion-weighted MRI

Background:

Non-invasive imaging biomarkers underpin the development of molecularly targeted anti-cancer drugs. This study evaluates tumour apparent diffusion coefficient (ADC), measured by diffusion-weighted magnetic resonance imaging (DW-MRI), as a biomarker of response to the MEK1/2 inhibitor selumetinib (AZD6244, ARRY-142886) in human tumour xenografts.

Methods:

Nude mice bearing human BRAF^V600D WM266.4 melanoma or BRAF^V600E Colo205 colon carcinoma xenografts were treated for 4 days with vehicle or selumetinib. DW-MRI was performed before and 2 h after the last dose and excised tumours analysed for levels of phospho-ERK1/2, cleaved caspase 3 (CC3) and necrosis.

Results:

Selumetinib treatment induced tumour stasis and reduced ERK1/2 phosphorylation in both WM266.4 and Colo205 tumour xenografts. Relative to day 0, mean tumour ADC was unchanged in the control groups but was significantly increased by up to 1.6-fold in selumetinib-treated WM266.4 and Colo205 tumours. Histological analysis revealed a significant increase in necrosis in selumetinib-treated WM266.4 and Colo205 xenografts and CC3 staining in selumetinib-treated Colo205 tumours relative to controls.

Conclusion:

Changes in ADC following treatment with the MEK1/2 inhibitor selumetinib in responsive human tumour xenografts were concomitant with induction of tumour cell death. ADC may provide a useful non-invasive pharmacodynamic biomarker for early clinical assessment of response to selumetinib and other MEK-ERK1/2 signalling-targeted therapies.     

High antitumor activity using intratumoral injection of plasmid DNA with mutant-type p27Kip1 gene following in vivo electroporation

In this study, we attempted to use a non-viral gene transfer system, in vivo electroporation, in oral cancer cell B88 xenografts. To evaluate this in vivo gene transfer method, the GFP gene was transfected into xenografts by electroporation. Then, the efficiency of transfection of exogenous p27Kip1 gene by electroporation was confirmed by Western blot analysis. Next, to estimate the reduction of oral cancer xenografts by this method, we measured the size of B88 xenografts in nude mice after electroporation with the wild- or mutant-type p27Kip1 gene. The growth of tumors was markedly suppressed by mutant-type p27Kip1 gene transfection by electroporation compared with transfection of wild-type p27Kip1 gene or empty vector only. Moreover, histological specimens revealed apoptotic cell death was increased in mutant-type p27Kip1-transfected tumors compared to wild-type or empty vector only. These results suggest that it is possible to transfer mutant-type p27Kip1 into oral cancer xenografts using electroporation and to suppress the growth of tumors, furthermore, it is suggested that this system might be used for oral cancer.     

Effect of 3,4-dihydro-6-[4-(3,4-dimethoxybenzoyl)-1-piperazinyl]-2(1H)-quinolinone (vesnarinone) on the growth of gastric cancer cell lines

Vesnarinone (OPC-8212; 3,4-dihydro-6-[4-(3,4-dimethoxybenzoyl)-1-piperazinyl]-2(1H)-quinolinone) is a synthetic oral cardiotonic agent that has been used for the treatment of patients with congestive heart failure. Six days of treatment with 30 μg/ml of vesnarinone induced 20–80% growth inhibitions in five out of six gastric carcinoma cell lines examined. Cell cycle analysis revealed that the vesnarinone-sensitive TMK-1 gastric cancer cell line exhibited a significant G0–G1 arrest without evidence of apoptotic cell death induction after 48 h of treatment. Interestingly, this phenomenon was preceded by a marked reduction in the expression of cyclin A, D1 and E as well as cyclin-dependent kinase 2 (CDK2). On the other hand, no significant change was observed in the expression of p21^Waf1/Cip1, p27^Kip1 nor various growth factors and their receptor genes. Overall these results indicate that vesnarinone inhibits the growth of gastric cancer cells by down-regulating G1 cyclins and CDK2 to induce G0–G1 arrest through a pathway different from that of cyclin inactivation by p21^Waf1/Cip1 or p27^Kip1.     

Role of p21 as a determinant of 1,6-Bis[4-(4-amino-3-hydroxyphenoxy)phenyl] diamantane response in human HCT-116 colon carcinoma cells

1,6-Bis[4-(4-amino-3-hydroxyphenoxy)phenyl]diamantane (DPD) induces growth inhibition in human cancer cells. In our previous study, we discovered that DPD irreversibly inhibits the growth of Colo 205 colon cancer cells at the G0/G1 phase and induces cell differentiation. However, the detailed mechanism is still unknown. In this study, we examined the functional importance of p21 and p53 in DPD-induced anticancer effects. We used three isogenic cell lines, HCT-116, HCT-116 p53-/- and HCT-116 p21-/-, to evaluate the roles of p21 and p53 in the in?vitro anticancer effects of DPD. The in?vivo anti-proliferative effect of DPD was demonstrated by HCT-116 and HCT-116 p21-/- xenograft models. DPD significantly inhibited the growth as well as increased the number of HCT-116 cells in the G0/G1 phase, but not in HCT-116 p53-/- and HCT-116 p21-/- cells examined by flow cytometry. Additionally, western blot analysis showed that DPD treatment induced p21, but not p53 protein expression in HCT-116 cells. The p21-associated cell cycle regulated proteins, such as cyclin D, CDK4 and pRb were decreased after DPD treatment in HCT-116 cells. The DPD-increased G0/G1 phase and induced cell cycle regulated protein expression were not observed in HCT-116 p21-/- and HCT-116 p53-/- cells. DPD decreased cell migration in HCT-116 and HCT-116 p53-/- but not in HCT-116 p21-/- cells. p21 was required for the DPD-induced in?vitro anti-colon cancer effect. The in?vivo study also showed that DPD significantly inhibited tumor growth through p21 signaling. Our results clearly demonstrate that DPD-induced in?vitro and in?vivo anticancer effects through the activation of p21 in HCT-116 cells.