8-Cl-adenosine-induced inhibition of colorectal cancer growth in vitro and in vivo

The cAMP analogue 8-Cl-cAMP induces apoptosis and inhibits proliferation of a wide variety of malignancies in vitro and in vivo with relatively little toxicity. The antitumor effects of this compound are thought to involve its ability to modulate type I protein kinase A (PKAI). However, a nontoxic metabolite of 8-Cl-cAMP, 8-Cl-adenosine, with no known activity against PKAI, exerts growth inhibitory effects in breast, ovary, pancreas, and colorectal cancer cells in vitro and accumulates in xenografted tumors after 8-Cl-cAMP treatment in vivo. To characterize further the antitumor effects of 8-Cl-adenosine in colorectal cancer, we examined its effects on cell growth in vitro (cell number, ³H-thymidine incorporation, and soft agar colony formation) using the isogenically matched colorectal cancer cell lines HCT116, HCT116-E6 (p53-depleted), and 80S14 (p21^WAF1/Cip1-null). 8-Cl-adenosine inhibited cell growth by 89%, 74%, and 79%, respectively in HCT116, HCT116-E6, and 80S14 cells after a 72-hour exposure. Growth inhibition coincided with DNA endoreduplication and subsequent apoptosis. Furthermore, nontoxic doses of 8-Cl-adenosine administered i.p. twice weekly for 4 weeks to athymic mice suppressed growth of HCT116-derived xenografts by 50%. These results show that 8-Cl-adenosine exerts antitumor activity against colorectal cancer independent of p53 and p21^WAF1/Cip1.     

Tumor suppression by resistant maltodextrin,Fibersol-2

Resistant maltodextrin Fibersol-2 is a soluble and fermentable dietary fiber that is Generally Recognized As Safe (GRAS) in the United States. We tested whether Fibersol-2 contains anti-tumor activity. Human colorectal cancer cell line, HCT116, and its isogenic cells were treated with FIbersol-2. Tumor growth and tumorigenesis were studied in vitro and in vivo. Apoptotic pathway and generation of reactive oxygen species (ROS) were investigated. We discovered that Fibersol-2 significantly inhibits tumor growth of HCT116 cells by inducing apoptosis. Fibersol-2 strongly induces mitochondrial ROS and Bax-dependent cleavage of caspase 3 and 9, which is shown by isogenic HCT116 variants. Fibersol-2 induces phosphorylation of Akt, mTOR in parental HCT116 cells, but not in HCT116 deficient for Bax or p53. It prevents growth of tumor xenograft without any apparent signs of toxicity in vivo. These results identify Fibersol-2 as a mechanism-based dietary supplement agent that could prevent colorectal cancer development.     

Emodin-Provoked Oxidative Stress Induces Apoptosis in Human Colon Cancer HCT116 Cells through a p53-Mitochondrial Apoptotic Pathway

Emodin, a natural anthraquinone isolated from the traditional Chinese medicine Radix rhizoma Rhei, can induce apoptosis in many kinds of cancer cells. This study demonstrated that emodin induces apoptosis in human colon cancer HCT116 cells by provoking oxidative stress, which subsequently triggers a p53-mitochondrial apoptotic pathway. Emodin induced mitochondrial transmembrane potential loss, increase in Bax and decrease in Bcl-2 expression and mitochondrial translocation and release of cytochrome c to cytosol in HCT116 cells. In response to emodin-treatment, ROS increased rapidly, and subsequently p53 was overexpressed. Pretreatment with the antioxidant NAC diminished apoptosis and p53 overexpression induced by emodin. Transfecting p53 siRNA also attenuated apoptosis induced by emodin, Bax expression and mitochondrial translocation being reduced compared to treatment with emodin alone. Taken together, these results indicate that ROS is a trigger of emodin-induced apoptosis in HCT116 cells, and p53 expression increases under oxidative stress, leading toBax-mediated mitochondrial apoptosis.     

Combinatorial inhibition of Plk1 and PKCβ in cancer cells with different p53 status

PKCβ and Plk1 are fascinating targets in cancer therapy. Therefore, we combined Enzastaurin targeting PKCβ and SBE13 targeting Plk1 to test synergistic effects in cells with different p53 status. We analyzed cell proliferation and apoptosis induction, and did Western blot and FACScan analyses to examine the combined PKCβ and Plk1 inhibition. p53-wild-type cells are more resistant to the combinatorial treatment than p53-deficient cells, which displayed a synergistic reduction of cell proliferation after the combination. HeLa, MCF-7 and HCT116^p53wt and HCT116^p53-/- cells differed in their cell cycle distribution after combinatorial treatment in dependence on a functional p53-dependent G1/S checkpoint (p53-deficient cells showed an enrichment in S and G2/M, p53-wild-type cells in G0/G1 phase). hTERT-RPE1 cells did not show the synergistic effects of cancer cells.Thus, we demonstrate for the first time that Plk1 inhibition using SBE13 enhances the effects of Enzastaurin in cancer cells. HCT116^p53wt and HCT116^p53-/- cells confirmed the p53-dependence of different effects after Plk1 and PKCβ inhibition observed in HeLa and MCF-7 cells. Obviously, p53 protects cells from the cytotoxicity of Enzastaurin in combination with SBE13. For that reason this combination can be useful to treat p53-deficient cancers, without displaying toxicity to normal cells, which all have functional p53.     

Exosomal microRNAs derived from colon cancer cells promote tumor progression by suppressing fibroblast TP53 expression

The tumor microenvironment offers favorable conditions for tumor progression, and activated fibroblasts, known as cancer‐associated fibroblasts, play a pivotal role. TP53‐deficient cancer cells are known to induce strong fibroblast activation. We aimed to elucidate the oncogenic role of exosomes derived from TP53‐deficient colon cancer cells in fibroblast proliferation and tumor growth. Cancer cell‐derived exosomes (CDEs) were isolated from the conditioned media of cancer cells using a sequential ultracentrifugation method. The effects of exosomes on tumor growth were evaluated using human cell lines (TP53‐WT colon cancer, HCT116; TP53‐mutant colon cancer, HT29; and fibroblasts, CCD‐18Co and WI‐38) and an immune‐deficient nude mouse xenograft model. HCT116 (HCT116^{sh p53}) cells deficient in TP53 accelerated cocultured fibroblast proliferation compared to TP53‐WT HCT116 (HCT116^{sh control}) cells in vitro. Exosomes from HCT116^{sh p53} cells suppressed TP53 expression of fibroblasts and promoted their proliferation. Xenografts of HCT116^{sh p53} cells grew significantly faster than those of HCT116^{sh control} cells in the presence of co‐injected fibroblasts, but this difference was diminished by CDE inhibition. Microarray analysis identified upregulation of several microRNAs (miR‐1249‐5p, miR‐6737‐5p, and miR‐6819‐5p) in TP53‐deficient CDEs, which were functionally proven to suppress TP53 expression in fibroblasts. Exosomes derived from TP53‐mutant HT29 cells also suppressed TP53 expression in fibroblasts and accelerated their growth. The proliferative effect of HT29 on cocultured fibroblasts was diminished by inhibition of these miRNAs in fibroblasts. Our results suggest that CDEs play a pivotal role in tumor progression by fibroblast modification. Cancer cell‐derived exosomes might, therefore, represent a novel therapeutic target in colon cancer.     

MSH3 expression does not influence the sensitivity of colon cancer HCT116 cell line to oxaliplatin and poly(ADP-ribose) polymerase (PARP) inhibitor as monotherapy or in combination

Purpose

Defective expression of the mismatch repair protein MSH3 is frequently detected in colon cancer, and down-regulation of its expression was found to decrease sensitivity to platinum compounds or poly(ADP-ribose) polymerase inhibitors (PARPi) monotherapy. We have investigated whether MSH3 transfection in MSH3-deficient colon cancer cells confers resistance to oxaliplatin or PARPi and whether their combination restores chemosensitivity.

Methods

MSH3-deficient/MLH1-proficient colon cancer HCT116^MLH1 cells were transfected with the MSH3 cDNA cloned into the pcDNA3.1(?) vector. MSH3/MLH1-deficient HCT116, carrying MLH1 and MSH3 mutations on chromosome 3 and 5, respectively, and HCT116 in which wild-type MLH1 (HCT116+3), MSH3 (HCT116+5) or both genes (HCT116+3+5) were introduced by chromosome transfer were also tested. Sensitivity to oxaliplatin and to PARPi was evaluated by analysis of clonogenic survival, cell proliferation, apoptosis and cell cycle.

Results

MSH3 transfection in HCT116 cells did not confer resistance to oxaliplatin or PARPi monotherapy. MSH3-proficient HCT116+5 or HCT116+3+5 cells, which were more resistant to oxaliplatin and PARPi in comparison with their MSH3-deficient counterparts, expressed higher levels of the nucleotide excision repair ERCC1 and XPF proteins, involved in the resistance to platinum compounds, and lower PARP-1 levels. In all cases, PARPi increased sensitivity to oxaliplatin.

Conclusions

Restoring of MSH3 expression by cDNA transfection, rather than by chromosome transfer, did not affect colon cancer sensitivity to oxaliplatin or PARPi monotherapy; PARP-1 levels seemed to be more crucial for the outcome of PARPi monotherapy.     

Radiobiological evaluation of forward and inverse IMRT using different fractionations for head and neck tumours

Introduction

We have shown that the radio sensitizer DCQ enhances sensitivity of HCT116 human colon cancer cells to hypoxia. However, it is not known whether the p53 or p21 genes influence cellular response to DCQ. In this study, we used HCT116 that are either wildtype for p53 and p21, null for p53 or null for p21 to understand the role of these genes in DCQ toxicity.

Methods

HCT116 cells were exposed to DCQ and incubated under normoxia or hypoxia and the viability, colony forming ability, DNA damage and apoptotic responses of these cells was determined, in addition to the modulation of HIF-1α and of p53, p21, caspase-2, and of the ataxia telangiectasia mutated (ATM) target PIDD-C.

Results

DCQ decreased colony forming ability and viability of all HCT116 cells to a greater extent under hypoxia than normoxia and the p21^-/-cell line was most sensitive. Cells had different HIF-1α responses to hypoxia and/or drug treatment. In p53^+/+, DCQ significantly inhibited the hypoxia-induced increases in HIF-1α protein, in contrast to the absence of a significant HIF-1α increase or modulation by DCQ in p21^-/- cells. In p53^-/- cells, 10 μM DCQ significantly reduced HIF-1α expression, especially under hypoxia, despite the constitutive expression of this protein in control cells. Higher DCQ doses induced PreG₁-phase increase and apoptosis, however, lower doses caused mitotic catastrophe. In p53^+/+ cells, apoptosis correlated with the increased expression of the pro-apoptotic caspase-2 and inhibition of the pro-survival protein PIDD-C. Exposure of p53^+/+ cells to DCQ induced single strand breaks and triggered the activation of the nuclear kinase ATM by phosphorylation at Ser-1981 in all cell cycle phases. On the other hand, no drug toxicity to normal FHs74 Int human intestinal cell line was observed.

Conclusions

Collectively, our findings indicate that DCQ reduces the colony survival of HCT116 and induces apoptosis even in cells that are null for p53 or p21, which makes it a molecule of clinical significance, since many resistant colon tumors harbor mutations in p53.     

辛二酰苯胺异羟肟酸对结肠癌HCT116和SW480细胞增殖、周期和凋亡的影响

目的:研究辛二酰苯胺异羟肟酸(suberoylanilide hydroxamic acid,SAHA)对结肠癌细胞系HCT116和SW480增殖、周期和凋亡的影响,并对其分子作用机制进行初步探讨.方法:将不同浓度SAHA分别处理结肠癌HCT116和SW480细胞后,MTT法检测SAHA对HCT116和SW480细胞增殖的影响,流式细胞仪检测HCT116和SW480细胞周期和细胞凋亡率,罗丹明(rhodamine) 123和二氯二氢荧光素二乙酸酯(DCFH-DA)法检测HCT116和SW480细胞线粒体跨膜电位(Aψm)和活性氧(ROS)水平,Real-time PCR和Western blotting法检测乙酰化组蛋白3(Ac-H3)、p21、CyclinD1、Bax和Bcl-2的mRNA和蛋白的表达水平.结果:SAHA作用于HCT116和SW480细胞48 h后,细胞增殖被抑制、细胞周期G1期比率升高、凋亡率升高(均P＜0.05),线粒体跨膜电位显著下降、细胞内ROS产生增多(均P＜0.05).与对照组比较,SAHA处理组p21和Bax mRNA增多、Cyclin D1和Bcl-2 mRNA表达量减少(均P＜0.05),相关蛋白Ac-H3、p21和Bax增多,CyclinD1和Bcl-2减少(均P＜0.05).结论:SAHA抑制结肠癌HCT116和SW480细胞增殖、阻滞细胞周期并诱导细胞凋亡,其机可能与调节p21、CyclinD1和Bcl-2家族基因的表达、促进组蛋白乙酰化有关.     

Ubiquitin ligase TRIM46 promotes the proliferation and invasion of colorectal cancer cells北大核心CSCD

Objective: To explore the effect of tripartite motif 46 (TRIM46) on the proliferation and invasion of colorectal cancer cells and study its possible mechanism. Methods: After TRIM46 siRNA was transfected into human colorectal cancer HCT116 cells, the expressions of TRIM46 mRNA and protein were detected by real-time fluorescent quantitative PCR and Western blotting, respectively. CCK-8 assay and Transwell chamber assay were implemented to observe the effect of TRIM46 down-regulation on the proliferation and invasion of HCT116 cells, and the expression of Src homology 2 (SH2)-containing tyrosine phosphatase 1 (SHP1) was detected by Western blotting. Results: After TRIM46 siRNA was transfected into human colorectal cancer HCT116 cells, the expression levels of TRIM46 mRNA and protein were inhibited (both P < 0.05). Knockdown of TRIM 46 significantly suppressed the cell proliferation and invasion of HCT116 cells (both P < 0.05). However, the expression level of SHP1 was increased (P < 0.05). Conclusion: Ubiquitin ligase TRIM46 may improve the proliferation and invasion of human colorectal cancer cells through suppressing SHP1. Copyright © 2017 by TUMOR All rights reserved.     

Loss of XIAP sensitizes colon cancer cells to PPARγ independent antitumor effects of troglitazone and 15-PGJ2

We investigated whether the anticancer effect of a combination of XIAP down-regulation and PPAR γ activation on colon cancer is PPARγ receptor dependent. HCT116-XIAP^+/+ cells and HCT116-XIAP^−/− cells were treated with troglitazone or 15-deoxy-Δ^12,14-prostaglandin J2 (15-PGJ2) with or without prior exposure to PPARγ inhibitor GW9662. Cell proliferation and apoptosis was evaluated. Athymic mice carrying HCT116-XIAP^−/− cells-derived tumors were treated with troglitazone in the presence or absence of GW9662. Inhibition of cell proliferation and induction of apoptosis by troglitazone and 15-PGJ2 were more prominent in HCT116-XIAP^−/− cells. PPARγ ligand-induced growth inhibition, apoptosis, caspase and PARP cleavage could not be blocked by GW9662. Troglitazone significantly retarded growth of xenograft tumors and this effect was not blocked by GW9662. Marked apoptosis and an up-regulation of E-cadherin were observed in xenograft tumor tissues, and GW9662 did not affect these effects. Thus, a combination of XIAP down-regulation and PPARγ ligands exert a significant anticancer effect in colon cancer via a PPARγ independent pathway.     

Procaine Inhibits the Proliferation and Migration of Colon Cancer Cells Through Inactivation of the ERK/MAPK/FAK Pathways by Regulation of RhoA

Colon cancer is one of the most lethal varieties of cancer. Chemotherapy remains as one of the principal treatment approaches for colon cancer. The anticancer activity of procaine (PCA), which is a local anesthetic drug, has been explored in different studies. In our study, we aimed to explore the anticancer effect of PCA on colon cancer and its underlying mechanism. The results showed that PCA significantly inhibited cell viability, increased the percentage of apoptotic cells, and decreased the expression level of RhoA in HCT116 cells in a dose-dependent manner (p < 0.05 or p < 0.01). Moreover, PCA increased the proportion of HCT116 cells in the G₁ phase as well as downregulated cyclin D1 and cyclin E expressions (p < 0.05). In addition, we found that PCA remarkably inhibited cell migration in HCT116 cells (p < 0.01). However, all these effects of PCA on cell proliferation, apoptosis, and migration were significantly reversed by PCA + pc-RhoA (p < 0.05 or p < 0.01). PCA also significantly decreased the levels of p-ERK, p-p38MAPK, and p-FAK, but PCA + pc-RhoA rescued these effects. Furthermore, the ERK inhibitor (PD098059), p38MAPK inhibitor (SB203580), and FAK inhibitor (Y15) reversed these results. These data indicate that PCA inhibited cell proliferation and migration but promoted apoptosis as well as inactivated the ERK/MAPK/FAK pathways by regulation of RhoA in HCT116 cells.     

Role of p53 and p21waf1/cip1 in senescence-like terminal proliferation arrest induced in human tumor cells by chemotherapeutic drugs.

Exposure of human tumor cell lines to moderate doses of anticancer agents induces terminal proliferation arrest accompanied by morphologic and enzymatic changes that resemble senescence of normal cells. We have investigated the role of p53 and p21waf1/cip1 in the induction of this response in drug-treated tumor cells. Doxorubicin treatment induced the senescence-like phenotype (SLP) and its associated terminal growth arrest in wild-type HCT116 colon carcinoma cells; this response was strongly decreased but not abolished in HCT116 lines with homozygous knockout of p53 or p21. Transduction of HT1080 fibrosarcoma cells with a genetic inhibitor of p53 also decreased the induction of SLP and increased drug-induced mitotic cell death. To determine if drug-stimulated p21 expression was responsible for senescence-like growth arrest, we have expressed different levels of p21 from an inducible promoter. While high-level overexpression of p21 was sufficient to induce SLP in HT1080 cells, the levels of p21 expressed in doxorubicin-treated cells could account for only a fraction of doxorubicin-induced SLP. Our results indicate that p53 and p21 act as positive regulators of senescence-like terminal proliferation arrest, but their function is neither sufficient nor absolutely required for this treatment response in tumor cells.     

Influence of p53 and p21Waf1 expression on G2/M phase arrest of colorectal carcinoma HCT116 cells to proteasome inhibitors

Ubiquitin-mediated protein degradation in vertebrates has been implicated in cell cycle control. In this report we explored the effects of proteasome inhibitors (MG132, lactacystin and ALLN) on cell cycle distribution. Colorectal carcinoma HCT116 cells were treated with proteasome inhibitor MG132. The results showed that MG132 inhibited cell proliferation in a dose-dependent manner. MG132 arrested HCT116 cells at G2/M phase, which was associated with drug-induced blockade of p53 degradation and/or induction of p53-related gene expression along with the accumulation of cyclin B, cyclin A and p21. MG132 treated HCT116 (wild-type) had a similar cell cycle distribution as the MG132 treated HCT116 (p53-/-) and HCT116 (p21-/-) cells, suggesting that p53 and p21 may not be essential for MG132-induced G2/M phase arrest. The release experiments from nocodazole-induced mitotic phase cells indicated that MG132 inhibits the proliferation of HCT116 cells via arrest in the G2 phase. In addition, when HCT116 cells were exposed to combination of sodium butyrate and MG132 enhanced cell growth inhibition and induction of apoptosis were observed.     

Cellular response and activation of apoptosis by mithramycin SK in p21-deficient HCT116 human colon carcinoma cells

HCT116 (p21^−/−) human colon carcinoma cells treated with mithramycin SK (MSK), a novel analog of the antitumor antibiotic mithramycin A (MTA), were transiently arrested in G2/M, with some cells entering a faulty mitotic cycle without cytokinesis that resulted in G1-like cell arrest, which consisted of post-mitotic aneuploid G1 cells. Some of these cells synthesized DNA and elicited an apoptotic response. The absence of p21^WAF1 made HCT116 cells more sensitive to MSK than to the related MTA. MSK also showed higher antiproliferative activity than MTA on HCT116 cells with different genetic backgrounds, including those lacking the p53 gene. Apoptosis in MSK-treated p21^−/− cells involved caspase 2 rather than caspase 3. Untreated HCT116 (p21^−/−) cells presented a little caspase 3 activity, which increased slightly after treatment with MSK. The apoptotic response in p21^−/− cells comprised caspase 2 acting as an executor caspase together with a loss of mitochondrial membrane potential that may be initiated by caspase 2. In contrast, caspase 3 was activated in wild-type HCT116 after treatment with MSK.     

Methanol Extract of Flacourtia indica Aerial Parts Induces Apoptosis via Generation of ROS and Activation of Caspases in Human Colon Cancer HCT116 Cells

Different plant parts of Flacourtia indica have long been used in Ayurvedic medicine. Previous studies have demonstrated that the methanolic extract of F. indica possess anti-inflammatory properties. The present study was aimed at investigating the anticancer effects of methanol extract of Flacourtia indica (FIM) aerial parts in human colon cancer (HCT116) cells. Treatment of cells with FIM at a concentration of 500 μg/ml for 24 hours significantly reduced cell viability and induced apoptosis, which was associated with the increased cytoplasmicexpression of cytochrome c, activation of caspase-3, and the cleavage of poly-(ADP-ribose) polymerase. Incubation with FIM also inhibited the levels of Bcl-2, Bcl-xl and survivin, which are the markers of cell proliferation, whereasthe expression of Bax remained unchanged. Treatment with FIM led to the generation of reactive oxygen species (ROS) in a concentration-dependent manner. Pharmacological inhibition of ROS generation by pretreatment ofcells with N-acetyl cysteine abrogated FIM-induced apoptosis in HCT116 cells. Thus, these results demonstrate that FIM has anti-proliferative and pro-apoptotic effects in HCT116 cells and the effects are, at least in part, due to the ROS dependent activation of caspases.     

Altered expression of cell proliferation-related and interferon-stimulated genes in colon cancer cells resistant to SN38

Irinotecan is a topoisomerase I inhibitor widely used as an anticancer agent in the treatment of metastatic colon cancer. However, its efficacy is often limited by the development of resistance. We have isolated a colon carcinoma cell line, HCT116-SN6, which displays a 6-fold higher resistance to SN38, the active metabolite of irinotecan. In this paper, we studied the molecular mechanisms that cause resistance to SN38 in the HCT116-SN6 cell line. First, we analyzed proliferation, cell cycle distribution, apoptosis, topoisomerase I expression and activity in SN38-resistant (HCT116-SN6) and sensitive (HCT116-s cells). We showed that the SN38-induced apoptosis and the SN38-activated cell cycle checkpoints leading to G(2)/M cell cycle arrest were similar in both cell lines. Topoisomerase I expression and catalytic activity were also unchanged. Then, we compared mRNA expression profiles in the two cell lines using the Affymetrix Human Genome GeneChip arrays U133A and B. Microarray analysis showed that among the genes, which were differentially expressed in HCT116-s and HCT116-SN6 cells, 27% were related to cell proliferation suggesting that proliferation might be the main target in the development of resistance to SN38. This result correlates with the phenotypic observation of a reduced growth rate in HCT116-SN6 resistant cells. Furthermore, 29% of the overexpressed genes were Interferon Stimulated Genes and we demonstrate that their overexpression is, at least partially, due to endogenous activation of the p38 MAP kinase pathway in SN38 resistant cells. In conclusion, a slower cell proliferation rate may be a major cause of acquired resistance to SN38 via a reduction of cell cycle progression through the S phase which is mandatory for the cytotoxic action of SN38. This lower growth rate could be due to the endogenous activation of p38.     

Mechanical deformation induces proliferation of human colorectal carcinoma cells

The cell biology of intravascular tumor cells is clinically important but the many important variables of this environment have proved difficult to model. We studied the effects of repetitive mechanical deformation, a phenomenon affecting all intravascular cells, on human colon cancer cell line HCT 116 in vitro. Cell proliferation, assessed by [3H]-thymidine incorporation and cell count, increased by about 30% at two days in cells subjected to deformation at 30 cycles/min as compared to controls; levels of the nuclear proliferation antigen detected by monoclonal antibody MIB-1 were also increased. Deformation increased transforming growth factor beta1 (TGF-beta1) and plasminogen activator inhibitor-1 gene expression sevenfold at two days, but mannose-6-phosphate did not affect cell proliferation, indicating that endogenous TGF-beta is not involved in the proliferative response. HCT 116 cells lack TGF-beta type II receptors, but stable transfection of TGF-beta type II receptor cDNA did not alter the cellular response to mechanical deformation, as assessed by cell proliferation, morphology, or gene expression. Mechanical deformation affects several important aspects of HCT 116 cell biology, suggesting that the intravascular environment may regulate tumor cell biology in general. Endogenous TGF-beta and TGF-beta receptor-mediated signaling are not responsible for the deformation-induced proliferative response in HCT 116.     

Polyploidy Formation in Doxorubicin-Treated Cancer Cells Can Favor Escape from Senescence

Cancer cells can undergo stress-induced premature senescence, which is considered to be a desirable outcome of anticancer treatment. However, the escape from senescence and cancer cell repopulation give rise to some doubts concerning the effectiveness of the senescence-induced anticancer therapy. Similarly, it is postulated that polyploidization of cancer cells is connected with disease relapse. We postulate that cancer cell polyploidization associated with senescence is the culprit of atypical cell divisions leading to cancer cell regrowth. Accordingly, we aimed to dissociate between these two phenomena. We induced senescence in HCT 116 cells by pulse treatment with doxorubicin and observed transiently increased ploidy, abnormal nuclear morphology, and various distributions of some proteins (e.g., p21, Ki-67, SA-β-galactosidase) in the subnuclei. Doxorubicin-treated HCT 116 cells displayed an increased production of reactive oxygen species (ROS) possibly caused by an increased amount of mitochondria, which are characterized by low membrane potential. A decrease in the level of ROS by Trolox partially protected the cells from polyploidization but not from senescence. Interestingly, a decreased level of ROS prevented the cells from escaping senescence. We also show that MCF7 cells senesce, but this is not accompanied by the increase of ploidy upon doxorubicin treatment. Moreover, they were stably growth arrested, thus proving that polyploidy but not senescence per se enables to regain the ability to proliferate. Our preliminary results indicate that the different propensity of the HCT 116 and MCF7 cells to increase ploidy upon cell senescence could be caused by a different level of the mTOR and/or Pim-1 kinases.     

p21(Waf1/Cip1) and p53 are downstream effectors of protein kinase C delta in tumor suppression and differentiation in human colon cancer cells

We have previously demonstrated that the delta isoform of protein kinase C (PKCdelta) is importantly involved in cell growth inhibition and tumor suppression in colon cancer cells. To investigate further the activity and mechanism of action of PKCdelta, we have retrovirally transduced a PKCdelta cDNA in HCT116 human colon cancer cells. PKCdelta-overexpressing cells (HCT116/PKCdelta) were growth-inhibited, showed marked morphologic changes and underwent multinucleation and phenotypic changes characteristic of mitotic catastrophe. Compared to controls, HCT116/PKCdelta cells showed a highly attenuated tumorigenic profile and poor anchorage-independent growth. In addition, transfected cells established junction-coordinated intercellular communications, expressed cell surface microvilli and overexpressed the colon differentiation marker alkaline phosphatase. HCT116/PKCdelta cells also produced the 89 kDa, carboxy-terminal catalytic domain of PARP. In HCT116/PKCdelta cells, p21(Waf1/Cip1) and p53 were transiently upregulated for 48 hr after PKCdelta transduction. In a p21 null subline of HCT116 cells (HCT116/p21null), overexpression of PKCdelta did not affect tumorigenicity or differentiation, indicating that p21 is essential for the antitumorigenic activity of PKCdelta. Similarly, overexpression of PKCdelta caused no significant phenotypic changes in HCT116/E6 cells, an HCT116 subline in which the p53 protein is downregulated by the human papillomavirus E6 gene product. We conclude that overexpression of PKCdelta in human colon cancer cells induces multiple antineoplastic effects that depend on the activities of p21(Waf1/Cip1) and p53.