Chemoresistant Colon Cancer Cells Research Articles

Downregulation of PHLPP Expression Contributes to Hypoxia-Induced Resistance to Chemotherapy in Colon Cancer Cells

Hypoxia is a feature of solid tumors. Most tumors are at least partially hypoxic. This hypoxic environment plays a critical role in promoting resistance to anticancer drugs. PHLPP, a novel family of Ser/Thr protein phosphatases, functions as a tumor suppressor in colon cancers. Here, we show that the expression of both PHLPP isoforms is negatively regulated by hypoxia/anoxia in colon cancer cells. Interestingly, a hypoxia-induced decrease of PHLPP expression is attenuated by knocking down HIF1α but not HIF2α. Whereas the mRNA levels of PHLPP are not significantly altered by oxygen deprivation, the reduction of PHLPP expression is caused by decreased protein translation downstream of mTOR and increased degradation. Specifically, hypoxia-induced downregulation of PHLPP is partially rescued in TSC2 or 4E-BP1 knockdown cells as the result of elevated mTOR activity and protein synthesis. Moreover, oxygen deprivation destabilizes PHLPP protein by decreasing the expression of USP46, a deubiquitinase of PHLPP. Functionally, downregulation of PHLPP contributes to hypoxia-induced chemoresistance in colon cancer cells. Taken together, we have identified hypoxia as a novel mechanism by which PHLPP is downregulated in colon cancer, and the expression of PHLPP may serve as a biomarker for better understanding of chemoresistance in cancer treatment.

Chronic Insulin Exposure does not Cause Insulin Resistance but is Associated with Chemo-resistance in Colon Cancer Cells

Insulin resistance is an adaptive process in insulin-sensitive tissues characterised by reduced insulin receptor (IR) and insulin-receptor substrate (IRS)-1 expression, increased IRS-1 serine phosphorylation and attenuated downstream signalling. We tested whether this molecular phenotype prevails in cancer cells after long-term exposure to insulin. We characterised expression of IR-related molecules, IRS-1 phosphorylation and downstream signalling in a panel of 5 colon cancer cell lines at different insulin exposures: 15 min (100 nM), approximating to acute stimulation; 48 h (100 nM), used to demonstrate adaptive changes; and 12 weeks (20 nM; chronic insulin exposure, CIE), approximating to chronic hyperinsulinaemia. To assess clinical relevance, we determined IC50 values (increased indicating chemo-resistance) in the CIE-treated cells using oxaliplatin, SN38 (irinotecan) and 5-fluorouracil (5-FU). All colon cancer cell lines (HCT 116, HT-29, C32, CaCo2, LoVo) were sensitive to 15 min insulin exposure with increased phosphorylation of Akt, PRAS40 and p70-S6K. At 48 h, there was incomplete or absent features of insulin resistance. In CIE-treated cells, there was reduced IR expression, incomplete IRS-1 adaptation, lack of signalling pathway attenuation and contra-adaptive increases in IRS-1 tyrosine phosphorylation in several cell types. In CIE cells, there were multiple examples of increased IC50 values (2- to 100-fold) following 24-h treatment with oxaliplatin and SN38, but not with 5-FU. We concluded that CIE in colon cancer cells does not completely induce an insulin resistance molecular phenotype but is associated with chemo-resistance. Adaptive changes seen in insulin-sensitive non-neoplastic cells in response to long-term insulin may not extrapolate to neoplastic cells.

Tu1593 Downregulation of miR-21 Induces Differentiation of Chemo-Resistant Colon Cancer Cells and Enhances Susceptibility to Therapeutic Regimens

Tu1593 Downregulation of miR-21 Induces Differentiation of Chemo-Resistant Colon Cancer Cells and Enhances Susceptibility to Therapeutic Regimens

Autophagy of cancer stem cells is involved with chemoresistance of colon cancer cells

Chemoresistance is a major cause of treatment failure in colon cancer, and cancer stem cells have been found to be involved in the chemoresistance of colon cancer. However, the mechanisms driving the chemoresistance of colon cancer stem cells have not been addressed. In this study, we investigated the cytotoxicity of paclitaxel in CD44(+)CD24(+) SW1222 colon cancer cells expressing Cdx1 (CD44(+)CD24(+)Cdx1(+) stem cells) and CD44(+)CD24(+) HCT116 colon cancer cells expressing wild-type p53 (CD44(+)CD24(+)p53wt stem cells). SW1222 cells were more resistant to paclitaxel-induced cytotoxicity than HCT116 cells. Conversely, HCT-116 cells had higher matrigel colony formation ability than SW1222 cells. The isolated CD44(+)CD24(+)Cdx1(+) cells showed higher resistance to paclitaxel-induced cytotoxicity than CD44(+)CD24(+)p53wt cells. The resistance of CD44(+)CD24(+)Cdx1(+) cells to paclitaxel is associated with upregulation of Cdx1 and Bcl-2 expression, caspase-3 activity, and the ratio of LC3-II/LC3-I. The sensitivity of CD44(+)CD24(+)p53wt cells to paclitaxel is associated with the downregulation of Bcl-2 expression, upregulation of Bax levels, and upregulation of caspase-3 activity. Silencing of Cdx1 expression and treatment with lysosomal inhibitor bafilomycin A increased paclitaxel-induced cytotoxicity in CD44(+)CD24(+)Cdx1(+) cells. Conversely, overexpression of Cdx1 decreased cell death in CD44(+)CD24(+)p53wt cells. Intratumoral injection of Cdx1 siRNA significantly inhibited tumor growth in a xenograft tumor model inoculated with CD44(+)CD24(+)Cdx1(+) cancer cells. Cdx1 exerts a protective role in colon cancer stem cells, which play a crucial role in chemoresistance to paclitaxel through activation of autophagy. Autophagy is activated though the Cdx1-Bcl-2-LC3 pathway. In contrast, p53 exerts a major role in apoptosis and inhibits autophagy in colon cancer stem cells.

Abstract 5204: Downregulation of PHLLP expression contributes to hypoxia-induced resistance to chemotherapy in colon cancer cells.

Abstract Hypoxia is a feature of solid tumors. Most tumors are at least partially hypoxic. This hypoxic environment plays a critical role in promoting resistance to anticancer drugs. PHLPP, a novel family of Ser/Thr protein phosphatases, functions as a tumor suppressor by negatively regulating Akt signaling in colon cancer. Here, we show that the expression of both PHLPP isoforms is negatively regulated by hypoxia in colon cancer cells. Hypoxia-induced decrease of PHLPP expression is attenuated by silencing HIF-1α but not HIF-2α, indicating that PHLPP downregulation is HIF-1α-dependent. Whereas the mRNA levels of PHLPP are not significantly altered in hypoxia, the reduction of PHLPP expression is caused by decreased protein translation downstream of mTOR and increased protein degradation. Specifically, hypoxia-induced downregulation of PHLPP is partially rescued in TSC2 or 4E-BP1 knockdown cells as the result of elevated levels of mTOR activity and protein synthesis. In addition, we show that hypoxia promotes PHLPP degradation by increasing the mRNA expression of USP46, a PHLPP-specific deubiquitinase, and resulting in upregulation of PHLPP ubiquitination. Functionally, downregulation of PHLPP plays an important role in hypoxia-induced chemoresistance in colon cancer cells. Taken together, we have identified hypoxia as a novel mechanism by which PHLPP is downregulated in colon cancer, and the expression of PHLPP may serve as a biomarker for better understand the drug resistance in cancer treatment. Citation Format: Yang-an Wen, Tianyan Gao. Downregulation of PHLLP expression contributes to hypoxia-induced resistance to chemotherapy in colon cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5204. doi:10.1158/1538-7445.AM2013-5204

Abstract 2077: Downregulation of miR-21 induces differentiation of chemo-resistant colon cancer cells and enhances susceptibility to therapeutic regimens.

Abstract MicroRNAs (miRNAs) are endogenous posttranscriptional modulators that negatively control the expression of their target genes and play an important role in the development and progression of colorectal carcinoma. In particular, microRNA-21 (miR-21), whose expression is greatly increased in chemotherapy-resistant (CR) colon cancer cells that are enriched in undifferentiated cancer stem/stem-like cells (CSCs/CSLCs). We hypothesize that miR-21 plays a critical role in regulating differentiation of colon cancer (CR cells). Indeed, we observed that downregulation of miR-21 in CR colon cancer HCT-116 cells by anti-sense-miR-21 induced differentiation, as evidenced by marked increases in CK-20 (cytokeratin-20) expression and alkaline phosphatase activity. These changes were accompanied by a significant reduction in the expression of colon CSC/CSLCs marker CD44, colonospheres formation and TCF/LEF activity and increased expression of the pro-apoptotic gene PDCD4. Induction of differentiation greatly increased the sensitivity of CR cells to the growth inhibitory effects of all three regimens tested: 5- FU+ Oxaliplatin (FUOX), difluorinated curcumin (CDF), an analog of curcumin and the combination of CDF and FUOX. However, the magnitude of inhibition of growth by either CDF (75%) alone or CDF + FUOX (80%) was much higher than that observed with FUOX (40%). Growth inhibition by CDF and CDF + FUOX in differentiating CR colon cancer cells was associated with a 98-99% reduction in the expression of CD44 and EGFR. The current observation suggest that CDF as well as CDF + FUOX are highly effective in inhibiting growth and reducing colon CSCs/CSLCs in anti-miR-21-induced differentiating CR colon cancer cells, which supports our contention that differentiating cells become susceptible to conventional and non-conventional therapeutic regimen. Citation Format: Yingjie Yu, Fazlul H. Sarkar, Adhip P.N. Majumdar. Downregulation of miR-21 induces differentiation of chemo-resistant colon cancer cells and enhances susceptibility to therapeutic regimens. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2077. doi:10.1158/1538-7445.AM2013-2077

Down-regulation of miR-21 Induces Differentiation of Chemoresistant Colon Cancer Cells and Enhances Susceptibility to Therapeutic Regimens

MicroRNAs are endogenous posttranscriptional modulators that negatively control the expression of their target genes and play an important role in the development and progression of many malignancies, including colorectal carcinoma. In particular, expression of microRNA-21 (miR-21) is greatly increased in chemotherapy-resistant (CR) colon cancer cells that are enriched in undifferentiated cancer stem/stem-like cells (CSCs/CSLCs). We hypothesize that miR-21 plays a critical role in regulating differentiation of CR colon cancer cells. Indeed, we observed that downregulation of miR-21 in CR colon cancer cells (HCT-116 or HT-29) by antisense miR-21 induced differentiation, as evidenced by marked increases in cytokeratin-20 (CK-20) expression and alkaline phosphatase activity. These changes were accompanied by a significant reduction in the expression of colon CSC/CSLC marker CD44, colonosphere formation, and T-cell factor/lymphoid enhancer factor (TCF/LEF) activity but increased the expression of proapoptotic programmed cell death 4 gene. Induction of differentiation greatly increased sensitivity of CR colon cancer cells to the growth inhibitory properties of all three regimens tested: 5-fluorouracil + oxaliplatin (FUOX), difluorinated curcumin (CDF), and the combination of CDF and FUOX. However, the magnitude of inhibition of growth by either CDF (75%) alone or CDF + FUOX (80%) was much higher than that observed with only FUOX (40%). Growth inhibition by CDF and CDF + FUOX in differentiating CR colon cancer cells was associated with a 98% to 99% reduction in the expression of CD44 and epidermal growth factor receptor (EGFR). However, down-regulation of CK-20 in CR colon cancer cells produced no significant change in cellular growth in the absence or presence of FUOX, when compared with the corresponding controls. The current observation suggests that CDF and CDF + FUOX are highly effective in inhibiting growth and reducing colon CSCs/CSLCs in anti-miR-21-induced differentiating CR colon cancer cells and supports our contention that differentiation enhances susceptibility of CR cancer cells to conventional and nonconventional therapeutic regimen.

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

ObjectiveDevelopment 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.MethodsWild 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.ResultsThe 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.ConclusionsCombining 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.

Expression of miR-34 is lost in colon cancer which can be re-expressed by a novel agent CDF

BackgroundColorectal Cancer (CRC) is one of the leading causes of death worldwide. Numerous cellular events, including deregulated expression of microRNAs (miRNAs), specifically the family of miR-34 consisting of miR-34a, b and c, is known to regulate the processes of growth and metastasis.MethodsWe evaluated the expression of miR-34 in formalin-fixed paraffin-embedded (FFPE) human colon cancer tissue specimens compared to normal colonic mucosa. Moreover, we also assessed the expression of miR-34 in colon cancer cell lines treated with our newly developed synthetic analogue of curcumin referred as difluorinated curcumin (CDF) compared to well known inhibitor of methyl transferase.ResultsWe found that the expression of miR-34a and miR-34c was down-regulated in colon cancer specimens compared to normal colonic mucosa and the loss of expression was also consistent with data from colon cancer cell lines. This down-regulation was attributed to promoter hypermethylation, because we found that the treatment of colon cancer cells with 5-aza-2´-deoxycytidine, a methyltransferase inhibitor, markedly induced the levels of miR-34a and miR-34c expression. Likewise, CDF was very effective in the re-expression of miR-34a and miR-34c, which was consistent with inhibition of cell growth of both chemo-sensitive and chemo-resistant colon cancer cells. The re-expression of miR-34 led to a marked reduction in the expression of its target gene, Notch-1.ConclusionThe loss of expression of miR-34 in colon cancer is in part due to promoter hypermethylation of miR-34, which can be re-expressed with our novel agent CDF, suggesting that CDF could be a novel demethylating agent for restoring the expression of miR-34 family, and thus CDF could become a newer therapeutic agent for the treatment of colon cancer.

Cisplatin-Induced Chemoresistance in Colon Cancer Cells Involves FXR-Dependent and FXR-Independent Up-Regulation of ABC Proteins

Export pumps often limit the usefulness of anticancer drugs. Here we investigated the effect of cisplatin on the expression of ABC proteins in human colon cancer cells. Short-term incubation of Caco-2 and LS174T cells with cisplatin resulted in up-regulation of several ABC pumps, in particular MRP2 and BCRP. In partially cisplatin-resistant cells (LS174T/R) obtained by long-term exposure to cisplatin, MRP2 and BCRP up-regulation was more marked. This was further enhanced when these cells were cultured under maintained stimulation with cisplatin. The MRP2 promoter (MRP2pr) was cloned, and partially deleted constructs linked to reporter genes were generated. Transfection of LS174T and LS174T/R cells with these constructs revealed the ability of cisplatin to activate MRP2pr. The intensity of this response was dependent on the conserved MRP2pr region. Basal MRP2pr activity was higher in LS174T/R cells, in which the expression of the transcription factors c/EBPβ, HNF1α, HNF3β, and HNF4α, but not PXR, p53, c-Myc, AP1, YB-1, NRF2, and RARα was enhanced. Up-regulation was particularly high for FXR (200-fold) and SHP (50-fold). In LS174T/R cells, GW4064 induced the expression of FGF19, SHP, OSTα/β, but not MRP2 and BCRP, although the sensitivity of these cells to cisplatin was further reduced. In LS174T cells, GW4064-induced chemoresistance was seen only after being transfected with FXR+RXR, when BCRP, but not MRP2, was up-regulated. Protection of LS174T cells against cisplatin was mimicked by transfection with BCRP. In conclusion, in colon cancer cells, cisplatin treatment enhances chemoresistance through FXR-dependent and FXR-independent mechanisms involving the expression of BCRP and MRP2, respectively.

Abstract 3937: SN-38 attenuates the chemoresistance acquired by colon cancer cells under hypoxic condition, via inhibition of hypoxia inducible factor (HIF) 1

Abstract Introduction: Hypoxia is a common feature of rapidly growing solid tumors. Under hypoxic condition, cancer cells acquire the resistance to chemotherapeutic agents. Accumulation of hypoxia-inducible factor (HIF)-1α in cancer cells plays important physiological roles, including resistance the therapy, through various mechanisms, including anti-apoptotic effects and the induction of G1 cell cycle arrest. Here, we aimed to clarify the mechanisms underlying the hypoxia-induced chemoresistance of colon cancer cells to 5-fluorouraacil (5-FU), oxaliplatin, and SN-38 (active metabolite of irinotecan), the standard agents for the chemotherapy of colorectal cancer. Methods: Human colon cancer cells, namely HT29 and SW480, were cultured under hypoxic condition (1% O2), and treated with the appropriate doses of the chemotherapeutics. The proliferative ability was analyzed by reading the fluorescence intensity of calcein-stained cells, and by counting the trypan blue-stained cells. Apoptosis was quantified by flow-cytometry after double-staining of the cells with AnnexinV/propidium iodide (PI). The cell cycle was evaluated by flow-cytometry after staining of cells with PI. HIF-1α expression was evaluated by Western blot analysis. Results: Under hypoxic condition, the proliferative ability of both HT29 and SW480 cells was significantly decreased compared with cells cultured under normoxia. Cells cultured under hypoxia were less responsive to 5-FU and oxaliplatin, compared to those cultured under normoxic condition, but were inhibited by SN-38 at the similar levels compared normoxia. Under hypoxia, a strong arrest of the cells to the G0/G1 phase was observed and, as a consequence, the typical changes of the cell cycle distribution, i.e., the intra-S arrest induced by 5FU and the G2/M arrest induced by oxaliplatin, were not identified after the treatment with the chemoterapeutics. By the treatment with SN-38, an increase of the fraction of sub-G1 phase cells was observed, which was confirmed to be dependent on the induction of apoptosis by the flow-cytometric analysis of AnnexinV-stained cells. SN-38 induced apoptosis of colon cancer cells at the similar rates under hypoxia and normoxia. Contrarily, the apoptosis rate induced by 5-FU and oxaliplatin was significantly reduced under hypoxia. Interestingly, SN-38 significantly reduced the expression of HIF-1α in colon cancer cells cultured under hypoxia, whereas neither 5-FU nor oxaliplatin affected its expression. Conclusion: Our findings suggest that SN-38 can inhibit the expression of HIF-1α in colon cancer cells under hypoxic condition, consequently overcoming the chemoresistance to this agent. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3937. doi:1538-7445.AM2012-3937

Abstract 3847: Lupeol, a novel inhibitor of Wnt/β-catenin signaling: Implications in colon cancer therapy

Abstract Introduction: Wnt/β-catenin signaling pathway which regulates a variety of cellular processes has been reported to play a critical role in the pathogenesis of human colon cancer and found in almost 90% of the colon cancer patients. Recent studies from our laboratory and others showed that Lupeol a natural triterpene acts as a potent and selective inhibitor of Wnt signaling in prostate and melanoma tumor cells (Saleem et al, Carcinogenesis, 30: 801-17, 2009). Keeping in view these reports, we hypothesized that Lupeol will inhibit the tumorigenic growth of colon cancer cells (exhibiting aberrant Wnt/α signaling) under in vitro and in vivo conditions. Objectives: We investigated the effect of Lupeol on growth of human normal colon and carcinoma cells (exhibiting varied Wnt profile) and determined if Lupeol could enhances the efficacy of Sulindac, (Wnt signaling inhibitor and chemotherapeutic agent) that is used in clinics to treat human colon cancer. Methods: To test our hypothesis we employed in vitro and in vivo strategies. Firstly, normal human colon epithelial cells (FHC) and colon carcinoma cell lines (SW-480, HCT-116, HT-29) treated with either Lupeol (10-50 μM), or Sulindac (100-200 μM), or combination (Lupeol + Sulindac) were tested for growth, proliferation, clonogenicity and surrogate biomarkers of Wnt signaling by employing 3[H]thymidine uptake, MTT, Soft-agar colony, FACS, immunoblot and luciferase-reporter assays. Secondly, we investigated whether Lupeol is bio-available to mice after oral and intraperitoneal (i.p.) administration by using mass spectrometry. Next, we treated athymic mice bearing colon tumors-derived from HT-29 cells (highly chemoresistant among all colon cells) with either Lupeol (40 mg/kg) or Sulindac (20 mg/kg) or combination and measured tumor growth. In vivo proliferation and biochemical analysis of harvested tumors for surrogate biomarkers of apoptosis, proliferation and Wnt signaling were measured by employing BrdU uptake, immunoblot and immunhistochemical analysis. Results: Lupeol treatment was observed to (i) inhibit the proliferation of colon cancer cells (exhibiting activated Wnt) while sparing normal cells, (ii) induce G0/G1 arrest and apoptosis, (iii) decrease cytoslic as well as nuclear β-catenin levels (iv) increase degradation of cytosolic β-catenin, (iv) decrease TCF-transcriptional activity and (v) decrease the expression of Wnt target genes. Lupeol was observed to bioavailable in mice after oral and i.p. administration. Lupeol significantly inhibited the tumorigenicity of colon tumor cells in a xenograft mouse model. Most importantly, Lupeol treatment significantly sensitized highly chemoresistant colon cancer cells to Sulindac therapy, in vitro and in vivo. Conclusion: Lupeol alone or as an adjuvant to current therapies could be developed as an agent to treat subtype of human colon cancer exhibiting constitutive activation of Wnt signaling. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3847. doi:1538-7445.AM2012-3847

Quantitative shape analysis of chemoresistant colon cancer cells: Correlation between morphotype and phenotype

Morphological, qualitative observations allow pathologists to correlate the shape the cells acquire with the progressive, underlying neoplastic transformation they are experienced. Cell morphology, indeed, roughly scales with malignancy.A quantitative parameter for characterizing complex irregular structures is the Normalized Bending Energy (NBE). NBE provides a global feature for shape characterization correspondent to the amount of energy needed to transform the specific shape under analysis into its lowest energy state.We hypothesized that a chemotherapy resistant cancer cell line would experience a significant change in its shape, and that such a modification might be quantified by means of NBE parameterization. We checked out the usefulness of a mathematical algorithm to distinguish wild and 5-fluorouracil (5-FU)-resistant colon cancer HCT-8 cells (HCT-8FUres). NBE values, as well as cellular and molecular parameters, were recorded in both cell populations.Results demonstrated that acquisition of drug resistance is accompanied by statistically significant morphological changes in cell membrane, as well as in biological parameters. Namely, NBE increased progressively meanwhile cells become more resistant to increasing 5-FU concentrations. These data indicate how tight the relationships between morphology and phenotype is, and they support the idea to follow a cell transition toward a drug-resistant phenotype by means of morphological monitoring.

Intracellular ATP Levels Are a Pivotal Determinant of Chemoresistance in Colon Cancer Cells

Altered metabolism in cancer cells is suspected to contribute to chemoresistance, but the precise mechanisms are unclear. Here, we show that intracellular ATP levels are a core determinant in the development of acquired cross-drug resistance of human colon cancer cells that harbor different genetic backgrounds. Drug-resistant cells were characterized by defective mitochondrial ATP production, elevated aerobic glycolysis, higher absolute levels of intracellular ATP, and enhanced HIF-1α-mediated signaling. Interestingly, direct delivery of ATP into cross-chemoresistant cells destabilized HIF-1α and inhibited glycolysis. Thus, drug-resistant cells exhibit a greater "ATP debt" defined as the extra amount of ATP needed to maintain homeostasis of survival pathways under genotoxic stress. Direct delivery of ATP was sufficient to render drug-sensitive cells drug resistant. Conversely, depleting ATP by cell treatment with an inhibitor of glycolysis, 3-bromopyruvate, was sufficient to sensitize cells cross-resistant to multiple chemotherapeutic drugs. In revealing that intracellular ATP levels are a core determinant of chemoresistance in colon cancer cells, our findings may offer a foundation for new improvements to colon cancer treatment.

Combination of dasatinib and curcumin eliminates chemo-resistant colon cancer cells

Metastatic colorectal cancer remains a serious health concern with poor patient survival. Although 5-Fluorouracil (5-FU) or 5-FU plus oxaliplatin (FOLFOX) is the standard therapy for colorectal cancer, it has met with limited success. Recurrence of the tumor after chemotherapy could partly be explained by the enrichment of the chemo-resistant sub-population of cancer stem cells (CSCs) that possess the ability for self-renewal and differentiation into different lineages in the tumor. Therefore development of therapeutic strategies that target CSCs for successful treatment of this malignancy is warranted. The current investigation was undertaken to examine the effectiveness of the combination therapy of dasatinib (a Src inhibitor) and curcumin (a dietary agent with pleiotropic effect) in inhibiting the growth and other properties of carcinogenesis of chemo-resistant colon cancer cells that are enriched in CSCs sub-population. Remnants of spontaneous adenomas from APCMin +/- mice treated with dasatinib and/or curcumin were analyzed for several cancer stem cell markers (ALDH, CD44, CD133 and CD166). Human colon cancer cells HCT-116 (p53 wild type; K-ras mutant) and HT-29 (p53 mutant; K-ras wild type) were used to generate FOLFOX resistant (referred to as CR) cells. The effectiveness of the combination therapy in inhibiting growth, invasive potential and stemness was examined in colon cancer CR cells. The residual tumors from APCMin +/- mice treated with dasatinib and/or curcumin showed 80-90% decrease in the expression of the CSC markers ALDH, CD44, CD133, CD166. The colon cancer CR cells showed a higher expression of CSCs markers, cell invasion potential and ability to form colonospheres, compared to the corresponding parental cells. The combination therapy of dasatinib and curcumin demonstrated synergistic interactions in CR HCT-116 and CR HT-29 cells, as determined by Calcusyn analysis. The combinatorial therapy inhibited cellular growth, invasion and colonosphere formation and also reduced CSC population as evidenced by the decreased expression of CSC specific markers: CD133, CD44, CD166 and ALDH. Our data suggest that the combination therapy of dasatinib and curcumin may be a therapeutic strategy for re-emergence of chemo-resistant colon cancer by targeting CSC sub-population.

Abstract 5381: A novel strategy to eliminate chemoresistant colon cancer cells

Abstract Recurrence of colon cancer, which affects nearly 50% of patients treated by conventional therapeutics, is thought to be due to re-emergence of chemotherapy-resistant cancer stem/stem-like cells (CSCs) Therefore, development of therapeutic strategies for targeted elimination of CSCs would be a novel treatment strategy for colon cancer. The current study examines whether diflourinated-curcumin (CDF), a novel analog of the dietary ingredient of curcumin, in combination with 5-fluorouracil and oxaliplatin (5-FU+Ox), the mainstay of colon cancer chemotherapeutic, would be an effective strategy to eliminate colon CSCs. Multiple molecular techniques that include real-time RT-PCR, Western blot analysis, MTT assay, caspase-3 activity, colonosphere formation in stem cell media, Hoechst-33342 dye exclusion and NF-κB-ELISA, were used. We observed that CDF together with 5-FU+Ox to be more potent than curcumin in reducing CD44 and CD166 expression. This was accompanied by inhibition of cell growth, induction of apoptosis and disintegration of colonospheres. These changes were associated with down-regulation of the membrane transporter protein ABCG2, and attenuation of EGFR, IGF-1R, and NF-κB signaling consistent with inactivation of β-catenin, COX-2, c-Myc and Bcl-xL and activation of the pro-apoptotic Bax. Our results suggest that CDF together with the conventional chemotherapeutics could be an effective treatment strategy for preventing the emergence of chemo-resistant colon cancer cells by eliminating CSCs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5381. doi:10.1158/1538-7445.AM2011-5381

Abstract 3503: Targeting chemo-resistant colon cancer cells by the combination of dasatinib and curcumin: A novel therapeutic strategy

Abstract Metastatic colorectal cancer remains a serious health concern with poor patient survival. 5-Fluorouracil (5-FU) or 5-FU plus oxaliplatin (FOLFOX), the standard therapy for colorectal cancer, has met with limited success. Recurrence of the tumor after chemotherapy could partly be explained by the enrichment of the chemo-resistant sub-population of cancer stem cells (CSCs) that possess the ability for self-renewal and differentiation into different lineages in the tumor. Therefore development of therapeutic strategy that target CSCs is highly desirable in reducing the risk of relapse and metastasis. The current investigation was undertaken to examine the effectiveness of the combination therapy of dasatinib (a Src inhibitor) and curcumin (a dietary ingredient) in inhibiting the growth and other properties of carcinogenesis of chemo-resistant colon cancer cells that are enriched in CSCs. Human colon cancer cells HCT-116 (p53 wild type; K-ras mutant) and HT-29 (p53 mutant; K-ras wild type) were used to generate FOLFOX resistant {referred to as chemoresistant (CR)} cells. The CR cells showed higher expression of CSCs markers, cell invasion potential and ability to form colonospheres, compared to the corresponding parental cells. The combination therapy of dasatinib and curcumin demonstrated synergistic interactions in CR-HCT-116 and CR-HT-29 cells. This caused inhibition of cellular growth, invasion and colonosphere formation and reduction in CSC population as evidenced by decreased expression of CSC specific markers: CD133, CD44, CD166 and ALDH. In conclusion, we propose that the combination therapy of dasatinib and curcumin may be an effective therapeutic strategy for re-emergence of chemo-resistant colon cancer by targeting CSC sub-population. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3503. doi:10.1158/1538-7445.AM2011-3503

Difluorinated-Curcumin (CDF): A Novel Curcumin Analog is a Potent Inhibitor of Colon Cancer Stem-Like Cells

Recurrence of colon cancer, which affects nearly 50% of patients treated by conventional therapeutics, is thought to be due to re-emergence of chemotherapy-resistant cancer stem/stem-like cells (CSCs). Therefore, development of therapeutic strategies for targeted elimination of CSCs would be a novel strategy. The current study examines whether difluorinated-curcumin (CDF), a novel analog of the dietary ingredient of curcumin, in combination with 5-fluorouracil and oxaliplatin (5-FU + Ox), the mainstay of colon cancer chemotherapeutic, would be effective in eliminating colon CSCs. Multiple methodologies that include real-time RT-PCR, Western blot, MTT assay, caspase-3 activity, colonosphere formation, Hoechst-33342 dye exclusion and NF-κB-ELISA were used. We observed that CDF together with 5-FU + Ox were more potent than curcumin in reducing CD44 and CD166 in chemo-resistant colon cancer cells, accompanied by inhibition of growth, induction of apoptosis and disintegration of colonospheres. These changes were associated with down-regulation of the membrane transporter ABCG2 and attenuation of EGFR, IGF-1R, and NF-κB signaling consistent with inactivation of β-catenin, COX-2, c-Myc and Bcl-xL and activation of the pro-apoptotic Bax. Our results suggest that CDF together with the conventional chemotherapeutics could be an effective treatment strategy for preventing the emergence of chemo-resistant colon cancer cells by eliminating CSCs.

Dose- and time-dependent effects of doxorubicin on cytotoxicity, cell cycle and apoptotic cell death in human colon cancer cells

The cytostatic drug doxorubicin is a well-known chemotherapeutic agent which is used in treatment of a wide variety of cancers. A key factor in the response of cancer cells to chemotherapeutic drugs is the activation of the apoptotic pathway, a pathway that is often impaired in chemoresistant colon cancer cells. The aim of the present study was to investigate the effects of doxorubicin in Hct-116 human colon carcinoma cells in order to clarify if a time/concentration range for optimal doxorubicin-induced apoptosis exists. We compared a treatment schedule were cells were bolus incubated for 3 h with doxorubicin followed by 24 h in drug-free medium, with a continuous doxorubicin treatment schedule for 24 h. Bolus incubation was carried out to determine effects of doxorubicin accumulated during the first 3 h, whereas continuous incubation allowed further (continuous) exposure to doxorubicin. We found that bolus (3 h) treatment with doxorubicin resulted in a dose-dependent decrease of viable cells and concomitant increase of apoptosis. Additionally, bolus (3 h) doxorubicin incubation led to phosphorylation of p53 at serine 392, induction of p21, G2 arrest and increase of proapoptotic protein Bax. In contrast, continuous (24 h) treatment with doxorubicin reduced the number of living cells with no parallel raise in the amount of dead cells. Continuous (24 h) treatment with 5 μM doxorubicin resulted in cell cycle arrest in G0/G1 phase that was neither accompanied by phosphorylation and activation of p53 nor enhanced expression of p21. These results suggest that doxorubicin is able to induce cell death by apoptosis only at particular dose and treatment conditions and imply a completely different cellular response following bolus or continuous exposure to doxorubicin.

Elimination of Colon Cancer Stem-Like Cells by the Combination of Curcumin and FOLFOX

5-Fluorouracil (5-FU) or 5-FU plus oxaliplatin (FOLFOX) remains the backbone of colorectal cancer chemotherapeutics but with limited success. This could partly be due to the enrichment of cancer stem cells (CSCs) that are resistant to conventional chemotherapy. Therefore, validation of a nontoxic agent that can either cause reversal of chemoresistance or promote the killing of CSCs would be highly desirable. The current study examines whether curcumin, the major active ingredient of turmeric, either alone or together with FOLFOX, would be an effective strategy to eliminate colon CSCs. Exposure of colon cancer HCT-116 or HT-29 cells to FOLFOX that inhibited their growth led to the enrichment of CSC phenotype as evidenced by increased proportion of CD133-, CD44-, and/or CD166-positive cells and epidermal growth factor receptor (EGFR) levels. Treatment of FOLFOX-surviving colon cancer cells with either curcumin alone or together with FOLFOX resulted in a marked reduction in CSCs, as evidenced by the decreased expression of CD44 and CD166 as well as EGFR and by their ability to form anchorage-dependent colonies. They also caused disintegration of colonospheres. Increased expression of EGFR in FOLFOX-surviving cells could be attributed to hypomethylation of the EGFR promoter, whereas an opposite phenomenon was observed when the FOLFOX-surviving cells were treated with curcumin and/or FOLFOX. These changes were accompanied by parallel alterations in the levels of DNA methyltransferase 1. In conclusion, our data suggest that curcumin by itself or together with the conventional chemotherapeutic could be an effective treatment strategy for preventing the emergence of chemoresistant colon cancer cells by reducing/eliminating CSCs.