Radiosensitization by Chir-124, a selective Chk1 inhibitor: Effects of p53 and cell cycle checkpoints

Checkpoint kinase-1 (CHK1) is a key regulator of the DNA damage-elicited G2-M checkpoints. The aim of the present study was to investigate the effects of a selective CHK1 inhibitor, Chir124, on cell survival and cell cycle progression following ionizing radiation (IR). Treatment with Chir-124 resulted in reduced clonogenic survival and abrogated the IR- induced G2-M arrest in a panel of isogenic HCT116 cell lines after IR. This radiosensitizing effect was relatively similar between p53-/- and p53-sufficient wild type (WT) HCT116 cells. However, the number of mitotic cells (as measured by assessing the phosphorylation of mitotic proteins) increased dramatically in p53-/- HCT116 cells after concomitant Chir-124 exposure, compared to IR alone, while no such effect was observed in p53-sufficient WT HCT116 cells. In p53-/- cells, Chir-124 treatment induced a marked accumulation of polyploid cells that were characterized by micronucleation or multinucleation. p21-/- HCT116 cells displayed a similar pattern of response as p53-/- cells. Chir-124 was able to radiosensitize HCT116 cells that lack checkpoint kinase-2 (CHK2) or that were deficient for the spindle checkpoint protein Mad2. Finally, Chir-124 could radiosensitize tetraploid cell lines, which were relatively resistant against DNA damaging agents. Altogether these results suggest that Chir-124-mediated radiosensitization is profoundly influenced by the p53 and cell cycle checkpoint system.     

Anticancer activity of a series of platinum complexes integrating demethylcantharidin with isomers of 1,2-diaminocyclohexane

A series of platinum complexes derived from integrating demethylcantharidin (DMC) with different isomers of 1,2-diaminocyclohexane (DACH) has been synthesized and found to exhibit superior in vitro anticancer activity against colorectal and human hepatocellular cancer cell lines when compared with oxaliplatin, cisplatin, and carboplatin. Flow cytometric analysis revealed that the trans-DACH-Pt-DMC analogues showed similar behavior to oxaliplatin on affecting the cell cycle of the HCT116 colorectal cancer cell line, but distinct from that of cisplatin or carboplatin. The DACH component apparently dictates the trans-DACH-Pt-DMC complexes to behave mechanistically similar to oxaliplatin, whereas the DMC ligand appears to enhance the compounds' overall anticancer activity, probably by accelerating the cell cycle from G1 to S-phase with subsequent onset of G2/M arrest and accompanying apoptosis.     

A Novel Aurora-A Inhibitor,BPR1K0609S1, Sensitizes Colorectal Tumor cells to 5-Fluorofracil (5-FU) Treatment

Small synthetic compounds have been implicated in treatment of human cancers. We have synthesized a small compound, BPR1K0609S1 (hereafter, BP), which inhibits Aurora-A kinase. In the present study, we studied the mechanism of BP suppression of tumorigenesis induced by Aurora-A. Given our previous results that inactivation of p53 accelerates MMTV-Aurora-A-mediated tumorigenesis in vivo, we studied the roles of p53 pathway using the isogenic human colon carcinoma cell lines of HCT116, in which p53, Puma, Bax, p21 or Chk2 is deleted. When these isogenic cell lines are treated with BP for 48 h, accumulation of G2M phase and aneuploidy are commonly observed, and HCT116 p21(-) cells show increase in apoptosis. In xenograft assay, s.c. injection of BP efficiently inhibits tumorigenesis of HCT116 deficient for Chk2 or p21. Re-transplantation of BP-resistant tumors indicates that these resistant cells do not acquire advanced tumor growth. Significantly, 5-FU (5-fluorouracil) treatment further induces apoptosis of BP-resistant HCT116 deficient for Chk2 or Puma. These results demonstrate that p21 deficiency enhances BP-mediated suppression of tumor growth, and that BP and 5-FU can collaborate for tumor regression.     

A novel microtubule depolymerizing colchicine analogue triggers apoptosis and autophagy in HCT‐116 colon cancer cells

Colchicine is a tubulin‐binding natural product isolated from Colchicum autumnale. Here we report the in vitro anticancer activity of C‐ring modified semi‐synthetic derivative of colchicine; N‐[(7S)‐1,2,3‐trimethoxy‐9‐oxo‐10‐(4‐phenyl‐piperidin‐1‐yl)‐5,6,7,9 tetrahydrobenzo[a]heptalen‐7‐yl]acetamide ( 4h ) on colon cancer HCT‐116 cell line. The compound 4h was screened for anti‐proliferative activity against different human cancer cell lines and was found to exhibit higher cytotoxicity against colon cancer cell lines HCT‐116 and Colo‐205 with IC₅₀ of 1 and 0.8 μM respectively. Cytotoxicity of the compound to the normal fR2 breast epithelial cells and normal HEK293 human embryonic kidney cells was evaluated in concentration and time‐dependent manner to estimate its selectivity for cancer cells which showed much better selectivity than that of colchicine. Compound 4h induced cell death in HCT‐116 cells by activating apoptosis and autophagy pathways. Autophagy inhibitor 3‐MA blocked the production of LC3‐II and reduced the cytotoxicity in response to 4h , but did not affect apoptosis, suggesting thereby that these two were independent events. Reactive oxygen species scavenger ascorbic acid pretreatment not only decreased the reactive oxygen species level but also reversed 4h induced cytotoxicity. Treatment with compound 4h depolymerized microtubules and the majority of cells arrested at the G2/M transition. Together, these data suggest that 4h has better selectivity and is a microtubule depolymerizer, which activates dual cell‐death machineries, and thus, it could be a potential novel therapeutic agent in cancer therapy. Copyright © 2016 John Wiley & Sons, Ltd.     

Selenium Compounds Activate ATM-dependent DNA Damage Response via the Mismatch Repair Protein hMLH1 in Colorectal Cancer Cells

Epidemiological and animal studies indicate that selenium supplementation suppresses risk of colorectal and other cancers. The majority of colorectal cancers are characterized by a defective DNA mismatch repair (MMR). Here, we have employed the MMR-deficient HCT 116 colorectal cancer cells and the MMR-proficient HCT 116 cells with hMLH1 complementation to investigate the role of hMLH1 in selenium-induced DNA damage response, a tumorigenesis barrier. The ATM (ataxia telangiectasia mutated) protein responds to clastogens and initiates DNA damage response. We show that hMLH1 complementation sensitizes HCT 116 cells to methylseleninic acid, methylselenocysteine, and sodium selenite via reactive oxygen species and facilitates the selenium-induced oxidative 8-oxoguanine damage, DNA breaks, G₂/M checkpoint response, and ATM pathway activation. Pretreatment of the hMLH1-complemented HCT 116 cells with the antioxidant N-acetylcysteine or 2,2,6,6-tetramethylpiperidine-1-oxyl or the ATM kinase inhibitor KU55933 suppresses hMLH1-dependent DNA damage response to selenium exposure. Selenium treatment stimulates the association between hMLH1 and hPMS2 proteins, a heterodimer critical for functional MMR, in a manner dependent on ATM and reactive oxygen species. Taken together, the results suggest a new role of selenium in mitigating tumorigenesis by targeting the MMR pathway, whereby the lack of hMLH1 renders the HCT 116 colorectal cancer cells resistant to selenium-induced DNA damage response.     

Methoxyamine potentiates DNA single strand breaks and double strand breaks induced by temozolomide in colon cancer cells

We have previously shown that human cancer cells deficient in DNA mismatch repair (MMR) are resistant to the chemotherapeutic methylating agent temozolomide (TMZ) and can be sensitized by the base excision repair (BER) blocking agent methoxyamine (MX) [21]. To further characterize BER-mediated repair responses to methylating agent-induced DNA damage, we have now evaluated the effect of MX on TMZ-induced DNA single strand breaks (SSB) by alkaline elution and DNA double strand breaks (DSB) by pulsed field gel electrophoresis in SW480 (O6-alkylguanine-DNA-alkyltransferase [AGT]+, MMR wild type) and HCT116 (AGT+, MMR deficient) colon cancer cells. SSB were evident in both cell lines after a 2-h exposure to equitoxic doses of temozolomide. MX significantly increased the number of TMZ-induced DNA-SSB in both cell lines. In contrast to SSB, TMZ-induced DNA-DSB were dependent on MMR status and were time-dependent. Levels of 50 kb double stranded DNA fragments in MMR proficient cells were increased after TMZ alone or in combination with O6-benzylguanine or MX, whereas, in MMR deficient HCT116 cells, only TMZ plus MX produced significant levels of DNA-DSB. Levels of AP endonuclease, XRCC1 and polymerase beta were present in both cell lines and were not significantly altered after MX and TMZ. However, cleavage of a 30-mer double strand substrate by SW480 and HCT116 crude cell extracts was inhibited by MX plus TMZ. Thus, MX potentiation of TMZ cytotoxicity may be explained by the persistence of apurinic/apyrimidinic (AP) sites not further processed due to the presence of MX. Furthermore, in MMR-deficient, TMZ-resistant HCT116 colon cancer cells, MX potentiates TMZ cytotoxicity through formation of large DS-DNA fragmentation and subsequent apoptotic signalling.     

Concentration- and stage-specific effects of nitrite on colon cancer cell lines

Colorectal cancer (CRC) is the second leading cause of cancer-related death in the United States. Nitrite in cured meats is thought to contribute to increased incidence of colon cancer. We sought to determine the effect of nitrite on human colon cancer cell lines at different stages. Our results indicate nitrite has no effect on proliferation of stage 1 SW116 colon cancer cells, while nitrite inhibits proliferation of stage 2 SW480 at 10 nM–100 μM and inhibits stage 3 HCT15 proliferation at 100 nM–1 μM, but promotes a significant increase in proliferation on stage 4 COLO205 cells at 100 μM. Furthermore, nitrite inhibited invasion into Matrigel® of stage 3 SW480 colon cancer cells in a concentration-dependent manner. However, it significantly promotes the invasion of stage 4 cells at 100 μM. Our FACS data demonstrated that nitrite decreased cell cycle progression in SW480 and HCT15 with arrested G2/M transition and delayed G1 phase entry in a concentration-dependent manner. However, 100 μM nitrite promoted cell cycle progression in COLO205 cells with increased S-phase entry. Taken together, our data indicate nitrite inhibits cancer cell progression at low concentrations and early stage but promotes cancer cell progression at higher concentrations in cells representing stage 4 colon carcinomas.     

DNA mismatch repair-dependent response to fluoropyrimidine-generated damage

Previous studies from our laboratory indicated that expression of the MLH1 DNA mismatch repair (MMR) gene was necessary to restore cytotoxicity and an efficient G(2) arrest in HCT116 human colon cancer cells, as well as Mlh1(-/-) murine embryonic fibroblasts, after treatment with 5-fluoro-2'-deoxyuridine (FdUrd). Here, we show that an identical phenomenon occurred when expression of MSH2, the other major MMR gene, was restored in HEC59 human endometrial carcinoma cells or was present in adenovirus E1A-immortalized Msh2(+/+) (compared with isogenic Msh2(-/-)) murine embryonic stem cells. Because MMR status had little effect on cellular responses (i.e. G(2) arrest and lethality) to the thymidylate synthase inhibitor, Tomudex, and a greater level of [(3)H]FdUrd incorporation into DNA was found in MMR-deficient cells, we concluded that the differential FdUrd cytotoxicity between MMR-competent and MMR-deficient cells was mediated at the level of DNA incorporation. Analyses of ATPase activation suggested that the hMSH2-hMSH6 heterodimer only recognized FdUrd moieties (as the base 5-fluorouracil (FU) in DNA) when mispaired with guanine, but not paired with adenine. Furthermore, analyses of incorporated FdUrd using methyl-CpG-binding domain 4 glycosylase indicated that there was more misincorporated FU:Gua in the DNA of MMR-deficient HCT116 cells. Our data provide the first demonstration that MMR specifically detects FU:Gua (in the first round of DNA replication), signaling a sustained G(2) arrest and lethality.     

Design,synthesis and biological evaluation of novel pteridinone derivatives possessing a hydrazone moiety as potent PLK1 inhibitors

A series of novel pteridinone derivatives possessing a hydrazone moiety were designed, synthesized and evaluated for their biological activity. Most of the synthesized compounds demonstrated moderate to excellent activity against A549, HCT116 and PC-3 cancer cell lines. In particular, compound L₁₉ exhibited the most potent antiproliferative effects on three cell lines with IC₅₀ values of 3.23 μM, 4.36 μM and 8.20 μM, respectively. In kinase assays, the compound L₁₉ also showed potent inhibition activity toward PLK1 with % inhibition values of 75.1. Further mechanism studies revealed that compound L₁₉ significantly inhibited proliferation of HCT-116 cell lines, induced a great decrease in mitochondrial membrane potential resulting in apoptosis of cancer cells, inhibited the migration of tumor cells, and arrested G1 phase of HCT116 cells.     

Synthesis and evaluation of novel α-substituted chalcones with potent anti-cancer activities and ability to overcome multidrug resistance

A series of forty α-substituted chalcones were synthesized and screened for their antiproliferative activities against HCT116 (colorectal) and HCC1954 (breast) cancer cell lines. Compounds 5a and 5e were found to be the most potent compounds with GI₅₀ values of 0.63 µM and 0.725 µM in HCC1954 cell line and 0.69 µM and 1.59 µM in HCT116 cell line, respectively. Both compounds induced a G2/M cell cycle arrest and caused apoptotic cell death in HCT116 cells as shown by the induction of PARP cleavage. The compounds also stabilized p53 in a dose-dependent manner in HCT116 cells following 24-hour treatment. Furthermore, both 5a and 5e were able to overcome multidrug resistance in two MDR-1 overexpressing multidrug resistant cell lines.     

Mismatch repair (MMR) efficiency and MSH2 gene mutation in human colorectal carcinoma cell line COLO320HSR

Colorectal cancer (CC) is one of two diseases, in which the link between cancer proneness and DNA repair deficiency appears to be proved. A strict relationship between mismatch repair (MMR) gene mutations, microsatellite instability (MSI) has been found in familiar colorectal cancer (Lynch syndrome). Tumorigenesis at familiar cancer is initiated by biallelic mutations in the major MMR genes, namely MSH2 or MLH1. One of these mutations is an inherited germline alteration and the other is a somatic one. The initiating mutation in sporadic colorectal tumors was not still identified although biochemical and genetic signs of MMR deficiency are observed in tumor cells. Two currently used colorectal tumor cell lines HCT116 and COLO320HSR were derived from hereditary and sporadic tumors accordingly. HCT116 cell line exhibits MMR-deficiency due to biallelic deletion in MLHL. As a consequence this shows MSI phenotype and a near-diploid karyotype. COLO320HSR cell line is characterized by MSS phenotype with mostly imbalanced aberrations. This indicates MMR proficiency in these cells. However, both MMR-deficient HCT116 and COLO320HSR cells reveal near-diploid karyotype. Earlier we have shown that the number of secondary DNA double strand breaks, induced by methylnitrosourea (MNU), represent functional activity of cellular MMR. In the present study, using this approach we evaluated sensitivity to MNU and MMR activity in two colorectal tumor cell lines (HCT 116, COLO320HSR) and compared them to that in the HeLa cell line, which have MMR-proficient phenotype. We showed that cell line COLO320HSR exhibits low MMR activity, close to the level of MMR-activity in HCT116 cell line. We found a mutation in MSH2-G520A gene in COLO320HSR. This neutral mutation apparently is not related to polymorphism as we failed to identify the same mutation in any of MSH2 gene sequences of lymphocytes from 30 patients with sporadic colorectal cancer.     

Saccharomyces cerevisiae lacking Snm1, Rev3 or Rad51 have a normal S-phase but arrest permanently in G2 after cisplatin treatment

The role of Snm1, Rev3 and Rad51 in S-phase after cisplatin (CDDP) DNA treatment has been examined. When isogenic deletion mutants snm1 delta, rev3 delta and rad51 delta were arrested in G1 and treated with doses of CDDP causing significant lethality (<20% survival in the mutant strains), they progressed through S-phase with normal kinetics. The mutants arrested in G2 like wild-type cells, however they did not exit the arrest and reenter the cell cycle. This finding demonstrates that these genes are not required to allow DNA replication in the presence of damage. Therefore, Snm1, Rev3 and Rad51 may act after S to allow repair. At high levels of damage (<40% survival in wild-type cells) S-phase was slowed in a MEC1-dependent fashion. The cross-link incision kinetics of snm1 delta and rev3 delta mutants were also examined; both showed no deficiencies in incision of cross-linked DNA.     

Comparison of geno- and cytotoxicity of methylnitrosourea on MMR-proficient and MMR-deficient human tumor cell lines

Deficient mismatch repair (MMR) is identified as a mutation of one of four major MMR genes and(or) microsatellite instability. These genomic changes are used as markers of MMR status of the heredity nonpolyposis colorectal cancer (HNPCC) spectrum tumors--familial and sporadic tumors of colon and extracolonic cancers fulfilling Amsterdam clinical criteria II. MMR-deficiency results in mutator phenotype and resistance to geno- and cytotoxicity of alkylating agents. The main cytotoxic damage to DNA in response to chemical methylation is O6-methylguanine (O6-mG). The secondary DNA strand breaks, which are formed during the MMR functioning, are proposed to be required for methylation induced cytotoxicity. We have assumed that the secondary double stand breaks (DSB) upon DNA methylation are able to represent functional efficiency of MMR in cells. The purpose of the paper was to test this assumption on human tumor cells differing in MMR-status and pulse-treated with methylnitrosourea (MNU). We used 3 cell lines: HeLa (MMR-competent endometrial tumor cells), HCT116 (MMR-deficient colorectal carcinoma cells), and Colo320 (sigmoid intestine tumor cells with uncharacterized MMR status). DSBs were evaluated with neutral comet assay. Cytotoxicity/viability was evaluated with MTT-asay and apoptotic index (frequency of morphologically determined apoptotic cells). We show that 1) cytotoxic effect of MNU (250 microM) on HeLa cells was exhibited 3 days after pulse-treatment of cells with MNU; 2) DSBs occurred 48 h after the drug treatment but prior to the onset of apoptosis of HeLa cells; 3) MMR-deficient HCT116 cells were resistant to the drug: no decreased viability, DSBs and apoptosis were observed during 3 days after cell treatment. Both cell lines exhibited high sensitivity to etoposide, classical inductor of unrepairable DSBs and p53. Etoposide has been found to induce DSBs in 6-12 h, which was followed by apoptosis (in 24 h). Colo320 cells exhibited intermediate position between HeLa and HCT116 cell lines in regard to sensitivity to MNU according to MTT-assay and the number of secondary DSBs formed in MNU-treated cells. Nevertheless, in contrast to HeLa cells, these breaks did not induce apoptosis in Colo320 cells. Our data confirm the assumption about case/effect relationship between secondary DNA double strand breaks, induced by monofunctional methylating agent MNU, and functioning of MMR in human tumor cells.     

Butyrate-induced apoptosis in HCT116 colorectal cancer cells includes induction of a cell stress response

Short chain fatty acids (SCFA), principally butyrate, propionate, and acetate, are produced in the gut through the fermentation of dietary fiber by the colonic microbiotica. Butyrate in particular is the preferred energy source for the cells in the colonic mucosa and has been demonstrated to induce apoptosis in colorectal cancer cell lines. We have used proteomics, specifically 2D-DIGE and mass spectrometry, to identify proteins involved in butyrate-induced apoptosis in HCT116 cells and also to identify proteins involved in the development of butyrate insensitivity in its derivative, the HCT116-BR cells. The HCT116-BR cell line was characterized as being less responsive to the apoptotic effects of butyrate in comparison to its parent cell line. Our analysis has revealed that butyrate likely induces a cellular stress response in HCT116 cells characterized by p38 MAPK activation and an endoplasmic reticulum (ER) stress response, resulting in caspase 3/7 activation and cell death. Adaptive cellular responses to stress-induced apoptosis in HCT116-BR cells may be responsible for the development of resistance to apoptosis in this cell line. We also report for the first time additional cellular processes altered by butyrate, such as heme biosynthesis and dysregulated expression of nuclear lamina proteins, which may be involved in the apoptotic response observed in these cell lines.     

Measurement of DNA mismatch repair activity in live cells

Loss of DNA mismatch repair (MMR) function leads to the development and progression of certain cancers. Currently, assays for DNA MMR activity involve the use of cell extracts and are technically challenging and costly. Here, we report a rapid, less labor-intensive method that can quantitatively measure MMR activity in live cells. A G–G or T–G mismatch was introduced into the ATG start codon of the enhanced green fluorescent protein (EGFP) gene. Repair of the G–G or T–G mismatch to G–C or T–A, respectively, in the heteroduplex plasmid generates a functional EGFP gene expression. The heteroduplex plasmid and a similarly constructed homoduplex plasmid were transfected in parallel into the same cell line and the number of green cells counted by flow cytometry. Relative EGFP expression was calculated as the total fluorescence intensity of cells transfected with the heteroduplex construct divided by that of cells transfected with the homoduplex construct. We have tested several cell lines from both MMR-deficient and MMR-proficient groups using this method, including a colon carcinoma cell line HCT116 with defective hMLH1 gene and a derivative complemented by transient transfection with hMLH1 cDNA. Results show that MMR-proficient cells have significantly higher EGFP expression than MMR-deficient cells, and that transient expression of hMLH1 alone can elevate MMR activity in HCT116 cells. This method is potentially useful in comparing and monitoring MMR activity in live cells under various growth conditions.     

Curcumin induces DNA damage and caffeine-insensitive cell cycle arrest in colorectal carcinoma HCT116 cells

Curcumin (CUR), a polyphenol derived from the plant Curcuma longa, displays potential anti-cancer activity. One of the mechanisms stems from its ability to elicit cell cycle arrest followed by suppression of cell proliferation. Herein, we reported that CUR significantly induced DNA damage and mediated S and G2/M phase arrest in colorectal carcinoma HCT116 cells. Unlike etoposide, a classical topoisomerase II inhibitor, CUR-triggered G2/M phase arrest was hardly reversed by caffeine (CAFF) which is an inhibitor of activated ataxia-telangiectasia-mutated (ATM)/ATM- and Rad3-related (ATR), indicating that ATM and ATR signaling pathways may be not involved in CUR-mediated S and G2/M phase arrest in HCT116 cells. Furthermore, we demonstrated that CUR caused mitosis arrest in HCT116 cells by using mitotic protein monoclonal antibody-2 as a mitosis marker and the surface plasmon resonance assay. The findings provide new mechanisms of cell proliferation inhibition triggered by CUR in HCT116 cells.