Association of the ERK1/2 and p38 kinase pathways with nitric oxide-induced apoptosis and cell cycle arrest in colon cancer cells

To investigate the mechanism by which nitric oxide (NO) induces cell death in colon cancer cells, we compared two types of colon cancer cells with different p53 status: HCT116 (p53 wild-type) cells and SW620 (p53-deficient) cells. We found that S-nitrosoglutathione (GSNO), the NO donor, induced apoptosis in both types of colon cancer cells. However, SW620 cells were much more susceptible than HCT116 cells to apoptotic death by NO. We investigated the role of extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 kinase on NO-induced apoptosis in both types of colon cancer cells. GSNO treatment effectively stimulated activation of the ERK1/2 and p38 kinase in both types of cells. In HCT116 cells, pretreatment with PD98059, an inhibitor of ERK1/2, or SB203580, an inhibitor of p38 kinase, had no marked effect on GSNO-induced apoptosis. However, in SW620 cells, SB203580 significantly reduced the NO-induced apoptosis, whereas PD098059 increases NO-induced apoptosis. Furthermore, we found evidence of cell cycle arrest of the G₀/G₁ phase in SW620 cells but not in HCT116 cells. Inhibition of ERK1/2 with PD098059, or of p38 kinase with SB203580, reduced the GSNO-induced cell cycle arrest of the G₀/G₁ phase in SW620 cells. We therefore conclude that NO-induced apoptosis in colon cancer cells is mediated by a p53-independent mechanism and that the pathways of ERK1/2 and p38 kinase are important in NO-induced apoptosis and in the cell cycle arrest of the G₀/G₁ phase.     

p53‐dependent G1 arrest in 1st or 2nd cell cycle may protect human cancer cells from cell death after treatment with ionizing radiation and Chk1 inhibitors

Objectives: This study was performed to explore the strategy of combining Chk1 inhibitors with ionizing radiation (IR) to selectively target p53‐deficient cancer cells. Materials and methods: Survival and cell cycle progression were measured in response to IR and the Chk1 inhibitors, UCN‐01 and CEP‐3891, in colon carcinoma HCT116 p53+/+ and p53?/? cells, and in osteosarcoma U2OS‐VP16 cells with conditional expression of dominant‐negative p53 (p53DD). Results: Clonogenic survival was selectively reduced in HCT116 p53?/? compared to p53+/+ cells after treatment with UCN‐01 and IR, and HCT116 p53+/+ cells also displayed strong p53‐dependent G₁ arrest in the 1st cell cycle after IR. In contrast, clonogenic survival was affected similarly in U2OS‐VP16 cells with and without expression of p53DD. However, death of U2OS‐VP16 cells was p53 dependent as assessed by cell viability assay at 72 h, and this was associated with p53‐dependent G₁ arrest in the 2nd cell cycle after treatment. Notably, HCT116 cells were overall more resistant than U2OS cells to cytotoxic effects of Chk1 inhibitors. Conclusion: Our results suggest that p53‐dependent G₁ arrest in both 1st and 2nd cell cycles may protect human cancer cells from cell death after treatment with IR and Chk1 inhibitors. However, a challenge for future clinical use will be that different cancers display different intrinsic sensitivity to such inhibitors.     

Houttuynia cordata Thunb extract modulates G0/G1 arrest and Fas/CD95-mediated death receptor apoptotic cell death in human lung cancer A549 cells

Background

Houttuynia cordata Thunb (HCT) is commonly used in Taiwan and other Asian countries as an anti-inflammatory, antibacterial and antiviral herbal medicine. In this study, we investigated the anti-human lung cancer activity and growth inhibition mechanisms of HCT in human lung cancer A549 cells.

Results

In order to investigate effects of HCT on A549 cells, MTT assay was used to evaluate cell viability. Flow cytometry was employed for cell cycle analysis, DAPI staining, and the Comet assay was used for DNA fragmentation and DNA condensation. Western blot analysis was used to analyze cell cycle and apoptotic related protein levels. HCT induced morphological changes including cell shrinkage and rounding. HCT increased the G₀/G₁ and Sub-G₁ cell (apoptosis) populations and HCT increased DNA fragmentation and DNA condensation as revealed by DAPI staining and the Comet assay. HCT induced activation of caspase-8 and caspase-3. Fas/CD95 protein levels were increased in HCT-treated A549 cells. The G₀/G₁ phase and apoptotic related protein levels of cyclin D1, cyclin A, CDK 4 and CDK 2 were decreased, and p27, caspase-8 and caspase-3 were increased in A549 cells after HCT treatment.

Conclusions

The results demonstrated that HCT-induced G₀/G₁ phase arrest and Fas/CD95-dependent apoptotic cell death in A549 cells     

Shogaols at proapoptotic concentrations induce G<Subscript>2</Subscript>/M arrest and aberrant mitotic cell death associated with tubulin aggregation

Shogaols have been previously reported to induce cancer cell death via multiple mechanisms, among which one analog 6-shogaol has been reported to cause microtubule damage through specific reaction with sulfhydryl groups in tubulin. In this study, a series of shogaols with different side chain lengths (4-, 6-, 8- and 10-shogaol) was synthesized and evaluated for antiproliferative activity in HCT 116 colon carcinoma and SH-SY5Y neuroblastoma cells. 4- and 6-shogaol were identified as lead compounds possessing the strongest antiproliferative activity. In the soft agar assay, the lead shogaols displayed dose-dependent inhibition on cancer cell colony formation under anchorage-independent conditions. Using HCT 116 as the selected cancer cell line, the molecular events linking shogaols-induced G₂/M cell cycle arrest to apoptosis characterized by caspase 3 and PARP cleavage were investigated. At sublethal concentrations, the halt at G₂/M phase was alleviated along time and cells survived. Conversely, proapoptotic concentrations of 4- and 6-shogaol induced irreversible G₂/M arrest that was at least in part associated with down-regulation of cell cycle checkpoint proteins cdk1, cyclin B and cdc25C, as well as spindle assembly checkpoint proteins mad2, cdc20 and survivin. A dose- and time-dependent accumulation of insoluble tubulin in the insoluble fractions of cell lysates provided evidence that G₂ checkpoint failure led to disruption of microtubule turnover. In summary, our results conclude that shogaols cause apoptosis by inducing aberrant mitosis at least through the attenuation of cell cycle and spindle assembly checkpoint proteins.     

4-O-methylhonokiol inhibits colon tumor growth via p21-mediated suppression of NF-κB activity

Biphenolic components in the Magnolia family have shown several pharmacological activities such as antitumor effects. This study investigated the effects of 4-O-methylhonokiol (MH), a constituent of Magnolia officinalis, on human colon cancer cell growth and its action mechanism. 4-O-methylhonokiol (0–30 μM) decreased constitutive activated nuclear factor (NF)-κB DNA binding activity and inhibited growth of human colon (SW620 and HCT116) cancer cells. It also caused G₀–G₁ phase cell cycle arrest followed by an induction of apoptotic cell death. However, knockdown with small interfering RNA (siRNA) of p21 or transfection with cyclin D1/Cdk4 binding site-mutated p21 abrogated MH-induced cell growth inhibition, inhibition of NF-κB activity as well as expression of cyclin D1 and Cdk4. Conversely, inhibition of NF-κB with specific inhibitor or siRNA augmented MH-induced apoptotic cell death. 4-O-methylhonokiol inhibited tumor growth, NF-κB activity and expression of antiapoptotic proteins; however, it increased the expression of apoptotic proteins as well as p21 in xenograft nude mice bearing SW620 cancer cells. The present study reveals that MH causes p21-mediated human colon cancer cell growth inhibition through suppression of NF-κB and indicates that this compound by itself or in combination with other anticancer agents could be useful for the treatment of cancer.     

The depletion of DNA methyltransferase-1 and the epigenetic effects of 5-aza-2′deoxycytidine (decitabine) are differentially regulated by cell cycle progression

5-Aza-2′-deoxycytidine (decitabine) is a drug targeting the epigenetic abnormalities of tumors. The basis for its limited efficacy in solid tumors is unresolved, but may relate to their indolent growth, their p53 genotype or both. We report that the primary molecular mechanism of decitabine—depletion of DNA methyltransferase-1 following its “suicide” inactivation—is not absolutely associated with cell cycle progression in HCT 116 colon cancer cells, but is associated with their p53 genotype. Control experiments affirmed that the secondary molecular effects of decitabine on global and promoter-specific CpG methylation and MAGE-A1 mRNA expression were S-phase dependent, as expected. Secondary changes in CpG methylation occurred only in growing cells ∼24–48 h after decitabine treatment; these epigenetic changes coincided with p53 accumulation, an index of DNA damage. Conversely, primary depletion of DNA methyltransferase-1 began immediately after a single exposure to 300 nM decitabine and it progressed to completion within ∼8 h, even in confluent cells arrested in G₁ and G₂/M. Our results suggest that DNA repair and remodeling activity in arrested, confluent cells may be sufficient to support the primary molecular action of decitabine, while its secondary, epigenetic effects require cell cycle progression through S-phase.Key words: 5-aza-2′-deoxycytidine, decitabine, DNA methyltransferase-1, suicide inactivation, p53, S-phase, cell cycle     

Discovery of N-aryl-9-oxo-9H-fluorene-1-carboxamides as a new series of apoptosis inducers using a cell- and caspase-based high-throughput screening assay. 1. Structure–activity relationships of the carboxamide group

N-(2-Methylphenyl)-9-oxo-9H-fluorene-1-carboxamide (2a) was identified as a novel apoptosis inducer through our caspase- and cell-based high-throughput screening assay. Compound 2a was found to be active with sub-micromolar potencies for both caspase induction and growth inhibition in T47D human breast cancer, HCT116 human colon cancer, and SNU398 hepatocellular carcinoma cancer cells. It arrested HCT116 cells in G₂/M followed by apoptosis as assayed by the flow cytometry. Structure–activity relationship (SAR) studies of the carboxamide group identified the lead compound N-(2-(1H-pyrazol-1-yl)phenyl)-9-oxo-9H-fluorene-1-carboxamide (6s). Compound 6s, with increased aqueous solubility, was found to retain the broad activity in the caspase activation assay and in the cell growth inhibition assay with sub-micromolar EC₅₀ and GI₅₀ values in T47D, HCT116, and SNU398 cells, respectively.     

Tumor-targeting Salmonella typhimurium A1-R decoys quiescent cancer cells to cycle as visualized by FUCCI imaging and become sensitive to chemotherapy

Quiescent cancer cells are resistant to cytotoxic agents which target only proliferating cancer cells. Time-lapse imaging demonstrated that tumor-targeting Salmonella typhimurium A1-R (A1-R) decoyed cancer cells in monolayer culture and in tumor spheres to cycle from G₀/G₁ to S/G₂/M, as demonstrated by fluorescence ubiquitination-based cell cycle indicator (FUCCI) imaging. A1-R infection of FUCCI-expressing subcutaneous tumors growing in nude mice also decoyed quiescent cancer cells, which were the majority of the cells in the tumors, to cycle from G₀/G₁ to S/G₂/M, thereby making them sensitive to cytotoxic agents. The combination of A1-R and cisplatinum or paclitaxel reduced tumor size compared with A1-R monotherapy or cisplatinum or paclitaxel alone. The results of this study demonstrate that A1-R can decoy quiescent cancer cells to cycle to S/G₂/M and sensitize them to cytotoxic chemotherapy. These results suggest a new paradigm of bacterial-decoy chemotherapy of cancer.     

p21Waf1/Cip1 Inhibition of Cyclin E/Cdk2 Activity Prevents Endoreduplication after Mitotic Spindle Disruption

During a normal cell cycle, entry into S phase is dependent on completion of mitosis and subsequent activation of cyclin-dependent kinases (Cdks) in G₁. These events are monitored by checkpoint pathways. Recent studies and data presented herein show that after treatment with microtubule inhibitors (MTIs), cells deficient in the Cdk inhibitor p21^Waf1/Cip1 enter S phase with a ≥4N DNA content, a process known as endoreduplication, which results in polyploidy. To determine how p21 prevents MTI-induced endoreduplication, the G₁/S and G₂/M checkpoint pathways were examined in two isogenic cell systems: HCT116 p21^+/+ and p21^−/− cells and H1299 cells containing an inducible p21 expression vector (HIp21). Both HCT116 p21^−/− cells and noninduced HIp21 cells endoreduplicated after MTI treatment. Analysis of G₁-phase Cdk activities demonstrated that the induction of p21 inhibited endoreduplication through direct cyclin E/Cdk2 regulation. The kinetics of p21 inhibition of cyclin E/Cdk2 activity and binding to proliferating-cell nuclear antigen in HCT116 p21^+/+ cells paralleled the onset of endoreduplication in HCT116 p21^−/− cells. In contrast, loss of p21 did not lead to deregulated cyclin D1-dependent kinase activities, nor did p21 directly regulate cyclin B1/Cdc2 activity. Furthermore, we show that MTI-induced endoreduplication in p53-deficient HIp21 cells was due to levels of p21 protein below a threshold required for negative regulation of cyclin E/Cdk2, since ectopic expression of p21 restored cyclin E/Cdk2 regulation and prevented endoreduplication. Based on these findings, we propose that p21 plays an integral role in the checkpoint pathways that restrain normal cells from entering S phase after aberrant mitotic exit due to defects in microtubule dynamics.     

Induction of Tumor Cell Death through Targeting Tubulin and Evoking Dysregulation of Cell Cycle Regulatory Proteins by Multifunctional Cinnamaldehydes

Multifunctional trans-cinnamaldehyde (CA) and its analogs display anti-cancer properties, with 2-benzoyloxycinnamaldehyde (BCA) and 5-fluoro-2-hydroxycinnamaldehyde (FHCA) being identified as the ortho-substituted analogs that possess potent anti-tumor activities. In this study, BCA, FHCA and a novel analog 5-fluoro-2-benzoyloxycinnamaldehyde (FBCA), were demonstrated to decrease growth and colony formation of human colon-derived HCT 116 and mammary-derived MCF-7 carcinoma cells under non-adhesive conditions. The 2-benzoyloxy and 5-fluoro substituents rendered FBCA more potent than BCA and equipotent to FHCA. The cellular events by which these cinnamaldehydes caused G₂/M phase arrest and halted proliferation of HCT 116 cells were thereby investigated. Lack of significant accumulation of mitosis marker phospho-histone H3 in cinnamaldehyde-treated cells indicated that the analogs arrested cells in G₂ phase. G₂ arrest was brought about partly by cinnamaldehyde-mediated depletion of cell cycle proteins involved in regulating G₂ to M transition and spindle assembly, namely cdk1, cdc25C, mad2, cdc20 and survivin. Cyclin B1 levels were found to be increased, which in the absence of active cdk1, would fail to drive cells into M phase. Concentrations of cinnamaldehydes that brought about dysregulation of levels of cell cycle proteins also caused tubulin aggregation, as evident from immunodetection of dose-dependent tubulin accumulation in the insoluble cell lysate fractions. In a cell-free system, reduced biotin-conjugated iodoacetamide (BIAM) labeling of tubulin protein pretreated with cinnamaldehydes was indicative of drug interaction with the sulfhydryl groups in tubulin. In conclusion, cinnamaldehydes treatment at proapoptotic concentrations caused tubulin aggregation and dysegulation of cell cycle regulatory proteins cdk1 and cdc25C that contributed at least in part to arresting cells at G₂ phase, resulting in apoptotic cell death characterized by emergence of cleaved forms of caspase 3 and poly (ADP-ribose) polymerase (PARP). Results presented in this study have thus provided further insights into the intricate network of cellular events by which cinnamaldehydes induce tumor cell death.     

Genomic and tumor biological aspects of the anticancer nicotinamide phosphoribosyltransferase inhibitor FK866 in resistant human colorectal cancer cells

Cancer cells' resistance to drugs remains an important problem affecting cancer treatment strategies. We previously studied the nicotinamide phosphoribosyltransferase (NAMPT) inhibitor FK866's resistance mechanisms in the human colorectal cancer HCT116 cells. We established an acquired FK866-resistant cell line, HCT116R^FK866. In this study, we investigated gene mutations in parental HCT116 and HCT116R^FK866 cells using exome sequencing technology. The results indicated cluster genes related to NAD⁺ biosynthesis (including NAMPT), DNA repair, and ATP-binding cassette transporters were differentially altered in these cells. Interestingly, HCT116R^FK866 cells, which are resistant to other class NAMPT inhibitors, were more sensitive to the anticancer 5-fluorouracil and cisplatin and γ-ray irradiation compared to parental HCT116 cells. This higher sensitivity appears to cause a genetic change in the identified gene clusters by resistance to the NAMPT inhibitor FK866. Collectively, these novel findings provide a better understanding of anticancer candidate NAMPT inhibitors with regard to resistance mechanisms and cancer chemotherapy strategies.     

Antitumor effects of a novel benzonaphthofurandione derivative (8e) on the human colon cancer cells in vitro and in vivo through cell cycle arrest accompanied with the modulation of EGFR and mTOR signaling

Benzonaphthofurandione has been considered as an important class of naturally occurring and synthetic compounds having a variety of biological functions. In this study, we evaluated the antitumor effects of 3-[2-(dimethylamino)isopropoxy]-1-hydroxybenzo[b]naphtho[2,3-d]furan-6,11-dione (8e), a novel benzonaphthofurandione derivative, on the growth of colorectal cancer HCT 116 cells both in vitro culture and an in vivo animal model.Compound 8e exhibited the potential growth inhibition of the colon cancer cells in a concentration-dependent manner. The anti-proliferative activity of 8e was also associated with the induction of cell cycle arrest in the G₀/G₁ phase. The 8e-induced cell cycle arrest was well correlated with the suppression of cyclin-dependent kinase 2 (CDK2), CDK4, cyclin D1, cyclin E, c-Myc, and phosphorylated retinoblastoma protein (pRb). The tumor growth in xenograft nude mice bearing HCT 116 cells by compound 8e (10 mg/kg) also significantly inhibited without any overt toxicity. In addition, the down-regulation of epidermal growth factor receptor (EGFR), Akt, and mTOR signalings were associated with the anti-proliferative activity of compound 8e in colon cancer cells. Taken together, these findings suggested that cell cycle arrest and modulation of cell signal transduction pathways might be the plausible mechanisms of actions for the anti-proliferative activity of 8e, and thus 8e might be used as an effective chemotherapeutic agent in human colon cancer.     

Invading cancer cells are predominantly in G0/G1 resulting in chemoresistance demonstrated by real-time FUCCI imaging

Invasive cancer cells are a critical target in order to prevent metastasis. In the present report, we demonstrate real-time visualization of cell cycle kinetics of invading cancer cells in 3-dimensional (3D) Gelfoam® histoculture, which is in vivo-like. A fluorescence ubiquitination cell cycle indicator (FUCCI) whereby G₀/G₁ cells express a red fluorescent protein and S/G₂/M cells express a green fluorescent protein was used to determine the cell cycle position of invading and non-invading cells. With FUCCI 3D confocal imaging, we observed that cancer cells in G₀/G₁ phase in Gelfoam® histoculture migrated more rapidly and further than cancer cells in S/G₂/M phases. Cancer cells ceased migrating when they entered S/G₂/M phases and restarted migrating after cell division when the cells re-entered G₀/G₁. Migrating cancer cells also were resistant to cytotoxic chemotherapy, since they were preponderantly in G₀/G₁, where cytotoxic chemotherapy is not effective. The results of the present report suggest that novel therapy targeting G₀/G₁ cancer cells should be developed to prevent metastasis.     

Tumor-specific cell-cycle decoy by Salmonella typhimurium A1-R combined with tumor-selective cell-cycle trap by methioninase overcome tumor intrinsic chemoresistance as visualized by FUCCI imaging

We previously reported real-time monitoring of cell cycle dynamics of cancer cells throughout a live tumor intravitally using a fluorescence ubiquitination cell cycle indicator (FUCCI). Approximately 90% of cancer cells in the center and 80% of total cells of an established tumor are in G₀/G₁ phase. Longitudinal real-time FUCCI imaging demonstrated that cytotoxic agents killed only proliferating cancer cells at the surface and, in contrast, and had little effect on the quiescent cancer cells. Resistant quiescent cancer cells restarted cycling after the cessation of chemotherapy. Thus cytotoxic chemotherapy which targets cells in S/G₂/M, is mostly ineffective on solid tumors, but causes toxic side effects on tissues with high fractions of cycling cells, such as hair follicles, bone marrow and the intestinal lining. We have termed this phenomenon tumor intrinsic chemoresistance (TIC). We previously demonstrated that tumor-targeting Salmonella typhimurium A1-R (S. typhimurium A1-R) decoyed quiescent cancer cells in tumors to cycle from G₀/G₁ to S/G₂/M demonstrated by FUCCI imaging. We have also previously shown that when cancer cells were treated with recombinant methioninase (rMETase), the cancer cells were selectively trapped in S/G₂, shown by cell sorting as well as by FUCCI. In the present study, we show that sequential treatment of FUCCI-expressing stomach cancer MKN45 in vivo with S. typhimurium A1-R to decoy quiescent cancer cells to cycle, with subsequent rMETase to selectively trap the decoyed cancer cells in S/G₂ phase, followed by cisplatinum (CDDP) or paclitaxel (PTX) chemotherapy to kill the decoyed and trapped cancer cells completely prevented or regressed tumor growth. These results demonstrate the effectiveness of the praradigm of “decoy, trap and shoot” chemotherapy.     

The p53-p21WAF1 checkpoint pathway plays a protective role in preventing DNA rereplication induced by abrogation of FOXF1 function

We previously identified FOXF1 as a potential tumor suppressor gene with an essential role in preventing DNA rereplication to maintain genomic stability, which is frequently inactivated in breast cancer through the epigenetic mechanism. Here we further addressed the role of the p53-p21^WAF1 checkpoint pathway in DNA rereplication induced by silencing of FOXF1. Knockdown of FOXF1 by small interference RNA (siRNA) rendered colorectal p53-null and p21^WAF1-null HCT116 cancer cells more susceptible to rereplication and apoptosis than the wild-type parental cells. In parental HCT116 cells with a functional p53 checkpoint, the p53-p21^WAF1 checkpoint pathway was activated upon FOXF1 knockdown, which was concurrent with suppression of the CDK2-Rb cascade and induction of G₁ arrest. In contrast, these events were not observed in FOXF1-depleted HCT116-p53−/− and HCT116-p21−/− cells, indicating that the p53-dependent checkpoint function is vital for inhibiting CDK2 to induce G₁ arrest and protect cells from rereplication. The pharmacologic inhibitor (caffeine) of ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3 related (ATR) protein kinases abolished activation of the p53-p21^WAF1 pathway upon FOXF1 knockdown, suggesting that suppression of FOXF1 function triggered the ATM/ATR-mediated DNA damage response. Cosilencing of p53 by siRNA synergistically enhanced the effect of FOXF1 depletion on the stimulation of DNA rereplication and apoptosis in wild-type HCT116. Finally, we show that FOXF1 expression is predominantly silenced in breast and colorectal cancer cell lines with inactive p53. Our study demonstrated that the p53-p21^WAF1 checkpoint pathway is an intrinsically protective mechanism to prevent DNA rereplication induced by silencing of FOXF1.     

Curcumin Enhances the Effect of Chemotherapy against Colorectal Cancer Cells by Inhibition of NF-κB and Src Protein Kinase Signaling Pathways

Objective

Development of treatment resistance and adverse toxicity associated with classical chemotherapeutic agents highlights the need for safer and effective therapeutic approaches. Herein, we examined the effectiveness of a combination treatment regimen of 5-fluorouracil (5-FU) and curcumin in colorectal cancer (CRC) cells.

Methods

Wild type HCT116 cells and HCT116+ch3 cells (complemented with chromosome 3) were treated with curcumin and 5-FU in a time- and dose-dependent manner and evaluated by cell proliferation assays, DAPI staining, transmission electron microscopy, cell cycle analysis and immunoblotting for key signaling proteins.

Results

The individual IC₅₀ of curcumin and 5-FU were approximately 20 µM and 5 µM in HCT116 cells and 5 µM and 1 µM in HCT116+ch3 cells, respectively (p<0.05). Pretreatment with curcumin significantly reduced survival in both cells; HCT116+ch3 cells were considerably more sensitive to treatment with curcumin and/or 5-FU than wild-type HCT116 cells. The IC₅₀ values for combination treatment were approximately 5 µM and 1 µM in HCT116 and 5 µM and 0.1 µM in HCT116+ch3, respectively (p<0.05). Curcumin induced apoptosis in both cells by inducing mitochondrial degeneration and cytochrome c release. Cell cycle analysis revealed that the anti-proliferative effect of curcumin and/or 5-FU was preceded by accumulation of CRC cells in the S cell cycle phase and induction of apoptosis. Curcumin potentiated 5-FU-induced expression or cleavage of pro-apoptotic proteins (caspase-8, -9, -3, PARP and Bax), and down-regulated anti-apoptotic (Bcl-xL) and proliferative (cyclin D1) proteins. Although 5-FU activated NF-κB/PI-3K/Src pathway in CRC cells, this was down-regulated by curcumin treatment through inhibition of IκBα kinase activation and IκBα phosphorylation.

Conclusions

Combining curcumin with conventional chemotherapeutic agents such as 5-FU could provide more effective treatment strategies against chemoresistant colon cancer cells. The mechanisms involved may be mediated via NF-κB/PI-3K/Src pathways and NF-κB regulated gene products.     

Hydrogen sulfide induces human colon cancer cell proliferation: Role of Akt,ERK and p21

H₂S (hydrogen sulfide), regarded as the third gaseous transmitter, is implicated in ulcerative colitis and colorectal cancers. The present study investigates the effects of H₂S on cell proliferation in human colon cancer HCT 116 cells and SW480 cells. We identified the two key enzymes, CBS and CSE, for H₂S synthesis in HCT 116 cells. An exogenously administered H₂S donor NaHS induced cell proliferation in a concentration‐dependent manner, with optimal proliferative concentration at 200 μmol/l. NaHS administration increased Akt and ERK phosphorylation. Blockade of Akt and ERK activation attenuated NaHS‐induced cell proliferation. Cell‐cycle analysis showed that NaHS treatment for 6 h decreased the proportion of cells in G₀–G₁ phase and increased the proportion of cells in S phase. Protein expressions of Cyclin D1 and PCNA (proliferating cell nuclear antigen) were not altered, but the cyclin‐dependent kinase inhibitor p21^Waf1/Cip1 was inhibited significantly by NaHS treatment. NaHS significantly reduced NO metabolite levels. In conclusion, NaHS induced human colon cancer cell proliferation. This effect might be mediated by the increase of Akt and ERK phosphorylation and the decrease of p21^Waf1/Cip1 expression and NO production. The results suggested a role for H₂S in human colonic cancer development.     

miR-155-TP53INP1调节轴在结直肠癌化疗药物敏感性中的作用机制

摘要 目的：初步揭示miR-155通过靶向调节TP53INP1表达水平影响结直肠癌细胞对5-FU化疗敏感性。方法：将人结肠直肠癌细胞系HCT116进行培养,提取细胞总RNA后,采用miR-155逆转录特异性引物构建反转录体系进行PCR扩增,通过qRT-PCR检测miR-155在5-FU耐药细胞HCT116/FU及敏感细胞株HCT116中的表达情况;取对数生长期细胞,分别转染miR-155mimics、miR-155抑制剂、miR-155阴性对照后,采用CCK-8法检测miR-155对细胞5-FU药物敏感性的影响,双荧光素酶报告基因系统验证miR-155与TP53INP1的靶基因关系,Western blot检测miR-155对 TP53INP1表达的影响。结果：miR-155在HCT116 /Fu细胞中的表达量是HCT116细胞的7.25倍;在相同5-FU浓度时,HCT116+阴性对照的细胞生长抑制率均高于HCT116+mimics、半数抑制浓度显著低于HCT116+mimics,差异均具有统计学意义(P＜0.05);TP53INP1是miR-155的靶基因,能显著降低野生型TP53INP1 3''-UTR的荧光素酶活性;转染miR-155 mimics后,TP53INP1的相对表达量显著下降,转染miR-155抑制剂后,TP53INP1的相对表达量显著升高,差异均具有统计学意义(P＜0.05)。结论：miR-155水平升高使HCT116细胞对5-FU的敏感性降低,miR-155可能通过靶向调节TP53INP1的表达水平,从而影响结直肠癌细胞对5-FU的敏感性。     

Antitumor Activity of a 5-Hydroxy-1H-Pyrrol-2-(5H)-One-Based Synthetic Small Molecule In Vitro and In Vivo

Alternative chemo-reagents are in great demand because chemotherapy resistance is one of the major challenges in current cancer treatment. 5-hydoxy-1H-pyrrol-2-(5H)-one is an important N-heterocyclic scaffold that is present in natural products and medicinal chemistry. However, its antitumor activity has not been systematically explored. In this study, we screened a panel of 5-hydoxy-1H-pyrrol-2-(5H)-one derivatives and identified compound 1d as possessing strong anti-proliferative activity in multiple cancer cell lines. Cell cycle analysis revealed that 1d can induce S-phase cell cycle arrest and that HCT116 was sensitive to 1d-induced apoptosis. Further analysis indicated that 1d preferentially induced DNA damage and p53 activation in HCT116 cells and that 1d-induced apoptosis is partly dependent on p53. Furthermore, we showed that 1d significantly suppressed tumor growth in xenograft tumor models in vivo. Taken together, our results suggest that 5-hydoxy-1H-pyrrol-2-(5H)-one derivatives bear potential antitumor activity and that 1d is an effective agent for cancer treatment.