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Keywords:

  • curcumin;
  • oxaliplatin;
  • colorectal cancer;
  • drug combinations

Abstract

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Colorectal cancer remains a leading cause of cancer death worldwide, despite markedly improved response rates to current systemic therapies. Oxaliplatin either alone or incorporated into 5-fluorouracil/leucovorin regimes has resulted in increased survival rates, particularly with regards to metastatic colorectal carcinoma. The chemopreventive polyphenol curcumin, which is currently in clinical trial, has been advocated for use in colorectal cancer either singly or in combination with chemotherapeutic drugs. In this study, the antiproliferative capacity of both compounds was compared in HCEC (normal-derived), HT29 (p53 mutant adenocarcinoma) and HCT116 (p53wt adenocarcinoma) colorectal cell lines to determine whether effects were cell-type specific at pharmacologically achievable doses, and whether the combination resulted in enhanced efficacy. Both oxaliplatin and curcumin displayed marked antiproliferative capacity at therapeutic concentrations in the two tumor cell lines. Order of sensitivity to oxaliplatin was HCT116>HT29>HCEC, whereas order of sensitivity to curcumin was HT29>HCT116>HCEC. HCT116 cells underwent induction of G2/M arrest in response to both oxaliplatin (irreversible) and curcumin (reversible). Apoptosis was induced by both agents, and up to 16-fold induction of p53 protein was observed in response to the combination. Antiproliferative effects in HT29 cells were largely cell cycle independent, and were mediated by induction of apoptosis. Effects were greatly enhanced in both cell lines when agents were combined. This study provides further evidence that curcumin may be of use in therapeutic regimes directed against colorectal cancer, and suggests that in combination with oxaliplatin it may enhance efficacy of the latter in both p53wt and p53 mutant colorectal tumors. © 2007 Wiley-Liss, Inc.

Current therapy options for colorectal cancer involve the combination of a variety of chemotherapeutic drugs, more recently including the third generation platinum-based drug oxaliplatin (Eloxatin). The safety profile for oxaliplatin is better than that for other platinum based drugs such as cisplatin, exhibiting greater antiproliferative capacity in combination with 5-FU1 and efficacy against cisplatin resistant tumors.2, 3 However, one of the risks with any drug combination is that of increased toxicity.4 It is therefore of particular importance that further therapeutic regimes are investigated to enhance efficacy, whilst minimizing unwanted toxic side effects.

The effects of the chemopreventive agent curcumin (derived from turmeric) in vitro have been widely documented,5–7 with growing evidence that it may provide antitumor efficacy, particularly when targeted against colorectal cancer, either alone or in combination6, 8, 9 More recently, curcumin has entered into clinical trials and has been administered at doses of up to 3.6 g daily for 6 months without dose limiting toxicity.10, 11 Whilst phase I trials have demonstrated little systemic toxicity and provided crucial information regarding tissue disposition, evidence is still lacking as to how curcumin may functionally contribute to clinical regimes. Advantages of chemopreventive agents as potential therapeutics include their long history of use within the human populace, inexpensiveness, ready availability and low toxicity profile, all of which should facilitate their use within the clinic.

The mechanisms of action for oxaliplatin arise from its ability to form several reactive species in physiological solutions, which bind covalently to DNA giving rise to both inter- and intra-strand platinum DNA adducts, mainly formed between the N7 position of adjacent guanines or between adjacent adenines and guanines.2 This leads to disruption of essential cellular processes, and ultimately results in cell death via activation of apoptotic pathways associated with DNA damage.

Curcumin has many documented effects on signaling pathways leading to induction of cell cycle arrest and/or apoptosis. However, there are few mechanistic studies that directly compare antiproliferative effects of chemotherapeutic versus chemopreventive agents at pharmacologically achievable concentrations. This is essential to predict patient response to new combinations of drugs.

Diet-derived agents offer excellent potential for a low-toxicity addition to the chemotherapeutic repertoire, but have yet to be evaluated in combination with oxaliplatin-based chemotherapeutic regimes. In this study we assessed the antiproliferative capacity of oxaliplatin and curcumin both singly and in combination on a normal-derived colorectal epithelial cell line and 2 colorectal cancer cell lines, using clinically achievable concentrations. The primary aims were as follows; to determine whether the same clinically sought-after endpoints of efficacy occurred following treatment with the proposed therapeutic, curcumin; to determine whether toxicity was selective for tumor cells or dependent on phenotype; to assess the potential for enhanced efficacy when the 2 agents were combined.

Material and methods

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Oxaliplatin (Sigma, Dorset, UK) was supplied as a lyophilized powder, and made up immediately prior to use in 5% glucose. Curcumin was obtained from Sigma and reconstituted in DMSO. Antibodies against bcl-2, p21 and p53 were from Dako (Cambridgeshire, UK), bax and bcl-xl from BD Biosciences (Oxford, UK) total chk1, pcdc2/total cdc2 from Cell Signaling Technology (MA), survivin from Novus Biologicals (Cambridgeshire, UK), caspase 9 from MBL International (MA), actin, bid and cdc25c from Santa Cruz Biotechnology (CA) and the Annexin V/FITC kit was from Bender Medsystems (Vienna, Austria). The Caspase-Glo 3/7, 8 and 9 assays were provided by Promega (Southampton, UK).

HCEC (kindly provided by Nestec, Lausanne, Switzerland) were cultured in DMEM (Sigma) in flasks, which were pre-coated with a cell adhering medium (serum free DMEM, 0.0065% BSA w/v, 1 μg/ml collagen, 2.5 ug/ml fibronectin). HT29 cells (ATCC, Middlesex, UK) were cultured in DMEM, and HCT116 wild type (wt), HCT116 p21 and p53 knockouts (kindly provided by B. Vogelstein, John Hopkins University, MD) were grown in McCoys 5A medium (Invitrogen, Paisley, UK). All cell lines required the presence of foetal calf serum (Invitrogen) in the medium, to a final concentration of 10%.

Treatment of cells

Cells were treated with oxaliplatin or curcumin alone or in combination for times up to 144 hr. All treatments contained equivalent concentrations of DMSO, which did not exceed 0.1%.

Cell proliferation assay

Five thousand cells per well were seeded onto 12-well plates and treated with the appropriate concentration of agents for 24, 72 and 144 hr. Cells were harvested and counted using a Beckman Coulter Z2 coulter particle counter and size analyzer (Beckman Coulter, Buckinghamshire, UK).

Annexin V staining for apoptosis

This was based on the method described previously,12 and allowed determination of phosphatidylserine externalization occurring during the later stages of apoptosis.

Cell cycle analysis

This was based on the method described previously.13 In brief, 1 × 105 or 2.5 × 105 cells were plated onto 6-well plates, left to adhere overnight and then treated with appropriate concentrations of agents for 8, 24, 48 or 72 hr. Adherent cells were trypsinized, washed ×2 in PBS and resuspended in 200 μl PBS. Cells were fixed by the addition of 2 ml ice cold 70% ethanol, whilst vortexing vigorously and incubated at 4°C for a minimum of 2 hr. Cells were pelleted (600g for 10 min) and resuspended in 800 μl PBS, whereupon RNase and PI were added to final concentrations of 0.1 mg/ml and 5 μg/ml respectively. The cells were incubated at 4°C overnight before analysis of DNA content, using the Becton Dickinson FACScan apparatus and Cell Quest software, with subsequent data analysis performed using Modfit LT software.

Caspase 3/7, 8 and 9 activity

These assays were based on the method described in Howells et al.14 Caspase activities were measured following treatment with oxaliplatin and curcumin, using the Caspase-Glo 3/7, 8 and 9 assay kits supplied by Promega. In brief, cells were seeded at 1 × 104 on a white 96 well Viewplate (Packard–Perkin Elmer, Milano, Italy) and left overnight. Medium was removed and replaced with 50 μl fresh medium containing appropriate concentrations of the agents, and cells were incubated for appropriate times prior to the addition of 50 μl Caspase-Glo substrate. Following agitation, the plate was incubated at room temperature for 1 hr in the dark prior to reading luminescence on a Fluostar Optima (BMG Labtech, Offenburg, Germany). Luminescence was proportional to caspase activity and expressed as fold change from DMSO control.

Western blotting

Cells were seeded at between 1 × 106 and 2.5 × 106 onto 9 cm plates and treated with oxaliplatin or curcumin for times up to 48 hr. Treated cells were lysed and samples analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting, followed by visualization using enhanced chemiluminescence (Amersham Life Science, Little Chalfont, UK). Blots were scanned using the Syngene chemigenius II (Cambridge, UK) and quantified using the Genetools software.

Statistics

All results were analyzed using a one way ANOVA, followed by Fisher's post hoc test.

Results

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Induction of apoptosis by oxaliplatin

DNA damage induced by oxaliplatin treatment will ideally result in tumor cell death. We initially sought to determine the relative sensitivities of our chosen cell lines to oxaliplatin, using apoptosis as an endpoint for efficacy (Fig. 1).

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Figure 1. Levels of apoptosis determined by annexin V staining in the 3 cell lines following treatment with increasing concentrations of oxaliplatin at 48 hr. Black bars represent live, white bars represent apoptotic and grey bars represent necrotic populations. Chart shows mean (n = 4) and standard deviation,* represents significant difference from DMSO control (p < 0.05).

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Oxaliplatin-induced apoptosis (as determined by annexin V staining) was not detected to a significant level in any cell line at 24 hr (data not shown). Following a 48 hr treatment, HCEC exhibited the least sensitivity, undergoing a nonsignificant 5% increase in apoptosis and a 10% increase in necrosis with 50 μM treatment. The HCT116 cells showed the greatest sensitivity, undergoing a small increase in apoptosis even at 1 μM oxaliplatin. Following a 50 μM treatment for 48 hr, similar modest increases in levels of apoptotic (∼16%) and necrotic cells (∼14%) were observed in both the HT29 and HCT116 cells.

Effect of oxaliplatin on proteins associated with cell cycle arrest and apoptosis

Oxaliplatin-induced antiproliferative effects have been linked to alterations in several cell cycle and apoptotic proteins in response to DNA damage. The effect of oxaliplatin treatments on the expression of p21, p53, cdc2 and survivin was investigated at 24 and 48 hr (Fig. 2).

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Figure 2. Western blots of cell cycle/cell survival-related proteins in the 3 colon cell lines following a 24/48 hr treatment with increasing concentrations of oxaliplatin (n = 3).

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HCEC cells exhibited a small induction of p21 (24 hr), which was amplified at 48 hr, even though no changes to levels of p53 were observed. Levels of phospho-cdc2, cdc2 and survivin were decreased from 2.5 μM at the later time point. In the HT29 cell line, oxaliplatin failed to induce any significant change to levels of p21 (no expression), mutant p53 or cdc2. Survivin levels were significantly decreased from 2.5 μM at 48 hr only. HCT116 cells, the most sensitive to the effects of oxaliplatin, showed significant induction of p21 and p53 (from 1 μM) at both time points. A slight decrease in phospho-cdc2 levels was observed at 5–20 μM (24 hr) with virtual abolition of phospho-cdc2 levels at 48 hr reflecting the dramatic decrease in levels of total cdc2. Survivin was decreased from 1 μM at both 24 and 48 hr (significantly so at 48 hr), with effects greatly enhanced following the longer treatment time.

Five micromolar oxaliplatin, which equates approximately to the highest in vivo dose that could be achieved following current clinical regimes,15 caused minimal apoptosis even in the HCT116 cells following a 48 hr treatment. This dose was subsequently chosen for combination treatments, and to compare oxaliplatin-mediated effects with those instigated by pharmacologically achievable concentrations of curcumin.

Effect of oxaliplatin in combination with curcumin on cell proliferation

Inhibition of proliferation by both curcumin and 5 μM oxaliplatin alone was enhanced by their combination in both tumor cell lines (Fig. 3). HCEC cells showed no significant decrease in cell number following a 24 hr treatment in response to any treatment. By 144 hr, however, oxaliplatin induced a 1.6-fold decrease in cell number, and 5 and 10 μM curcumin a 1.4 and 3.8-fold decrease respectively. No significant further decrease was observed when oxaliplatin was combined with curcumin compared with either treatment alone. HT29 cells were similar to the HCEC in that cell number was not significantly altered following 24 hr treatments. Following the longer treatments however, their sensitivity was much more striking. Oxaliplatin alone inhibited proliferation by 3.6-fold after 144 hr treatment. Sensitivity to curcumin was also more marked, with proliferation inhibited by 15.3-fold following treatment with 1 μM. Oxaliplatin in combination with 1 μM curcumin resulted in a greatly enhanced inhibition of 87-fold compared with the DMSO control.

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Figure 3. Mean fold change in cell number (n = 4) from DMSO control in HCEC, HT29 and HCT116 cells following treatment with curcumin +/− oxaliplatin at 24 (hatched bars), 72 (grey bars) or 144 (black bars) hr +/− SEM. * represents significant difference from DMSO control, ‘a’ represents a significant difference from oxaliplatin alone, and ‘b’ represents a significant difference from the equivalent concentration of curcumin alone (p < 0.05).

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HCT116 cells have a very high proliferative index, and both oxaliplatin and curcumin were able to prevent proliferation (up to 3-fold) following a 24 hr treatment. At 144 hr, HCT116 cells exhibited the highest degree of sensitivity to oxaliplatin (16.8-fold inhibition), but showed markedly less sensitivity to the effects of curcumin (1.7-fold inhibition following 1 μM treatment) at the lower doses. However, following the combination of oxaliplatin with 1 μM curcumin, proliferation was decreased by 344-fold when compared with the DMSO control.

Induction of apoptosis following oxaliplatin and curcumin treatment

Induction of apoptosis was assessed via a variety of methods. Following a 24 hr treatment, caspase 8 cleavage was increased in both the HT29 and HCT116 cells by oxaliplatin alone, and the combination treatments (43, 41 and 18 kd fragments) (Fig. 4a). A slight increase in caspase 9 cleavage products were noted for the combination treatment in the HT29 cells, and for oxaliplatin and curcumin treatments in the HCT116 cells. However, no significant changes to any of the bcl-2-related proteins investigated were observed. Both caspase 8 and 9 activities were increased from 12 hr (Fig. 4b) following a treatment of either 5 μM oxaliplatin or 20 μM curcumin in both cell lines, with caspase 8 presenting as the dominant caspase.

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Figure 4. Apoptosis induced by curcumin +/− oxaliplatin in the colon tumor cell lines. (a) Western blots showing caspase cleavage and levels of bcl-2-related proteins in colon tumor cell lines following a 24 hr treatment with curcumin +/− oxaliplatin (n = 3). (b) Caspase 8 (black bars) and 9 (grey bars) activity in colon tumor cell lines following a time course with either 5 μM oxaliplatin or 20 μM curcumin. Chart shows mean (n = 6) and standard deviation, * represents significant difference from DMSO control for respective caspase. (c) Caspase 3/7 activity in 2 colon tumor cell lines following a 24 hr treatment with curcumin +/− oxaliplatin. Chart shows mean (n = 3) and standard deviation, * represents significant difference from DMSO control, “a” represents a significant difference from oxaliplatin alone, and “b” represents a significant difference from the equivalent concentration of curcumin alone (p < 0.05). (d) Levels of apoptosis in tumor cell lines determined by annexin V staining following a 48 hr treatment with curcumin +/− oxaliplatin. Black bars represent live, white bars represent apoptotic and grey bars represent necrotic populations. Chart shows mean (n = 3) and standard deviation, * represents a significant difference from DMSO control (p < 0.05).

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Effector caspase activity (3/7) was then measured at 24 hr in response to the combination treatments (Fig. 4c).

In HCEC cells, there was no significant induction of caspase activity with any treatment (data not shown). Caspase activity in the HT29 cells was not increased by 5 μM oxaliplatin or 5 μM curcumin, but was significantly induced at doses of 10 and 20 μM curcumin alone in a dose-dependent manner. When the treatments were combined, activity was increased 1.20-, 1.93- and 1.58-fold by 5, 10 and 20 μM curcumin when compared with curcumin treatments alone, and was significantly different from individual agent treatments. In the HCT116 cells a significant increase in caspase activity was observed with the higher doses of curcumin and was enhanced by combined treatments (1.29-, 1.47- and 2.68-fold compared with 5, 10 and 20 μM curcumin alone), which was again significantly different from both the oxaliplatin and equivalent curcumin treatments alone.

Caspase activity data were further corroborated using the annexin V binding assay at a slightly later time point (Fig. 4d).

Following 48 hr treatments, there was no significant induction of apoptosis in the HCEC cells (data not shown). In the HT29 cells, whereas oxaliplatin alone did not induce apoptosis at this time point, curcumin caused a dose-dependent increase in cell death, becoming significant at 20 μM. Cell death was enhanced when oxaliplatin was combined with the lower concentrations of curcumin; in particular, there was an increase in apoptosis of 10% compared with 5 μM curcumin alone, whilst the percentage of cells in the necrotic population remained at similar levels.

In the HCT116 cells, no significant induction of apoptosis occurred with oxaliplatin or at low doses of curcumin, while 20 μM curcumin increased apoptosis by 22%. A combination of oxaliplatin with 10 μM curcumin increased apoptotic cells compared with curcumin alone, but the combination of 20 μM curcumin plus oxaliplatin was similar to treatment with 20 μM curcumin alone.

Effect of oxaliplatin and curcumin on cell cycle distribution

Oxaliplatin and curcumin are known to cause G2/M cell cycle arrest in some cell lines,16–18 which may account for the decrease in proliferative capacity following treatments with these agents, both singly and combined in the 2 colon tumor cell lines.

Following a 24 hr treatment (Fig. 5a), HCEC underwent a significant G2/M arrest in response to 20 μM curcumin, which was not significantly increased following the combined treatment. At 48 hr, there was significant S-phase accumulation following oxaliplatin treatment, and curcumin treatment (20 μM) resulted in 53% of cells arresting in G2/M. Whilst the combined treatment with 10 μM curcumin resulted in a significant G2/M-accumulation compared with the DMSO control or equivalent concentration of curcumin alone, the combination of oxaliplatin with 20 μM curcumin abrogated the effect of this concentration of curcumin alone. By 72 hr, the effect of the agents individually was lost, whilst a combination of oxaliplatin with the lowest dose of curcumin resulted in significant G2/M arrest. This was again abrogated with increasing curcumin concentrations.

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Figure 5. Effect of curcumin +/− oxaliplatin on cell cycle events in the 3 colon cell lines. (a) Cell cycle distribution (%) in the 3 colon cell lines following a 24, 48 and 72 hr treatment with curcumin +/− oxaliplatin. Black bars represent the percentage of cells in G1, white bars the percentage of cells in S phase and grey bars the percentage of cells in G2/M. Chart shows mean (n = 3) and standard deviation, * represents significant difference from DMSO control, “a” represents a significant difference from oxaliplatin alone, and “b” represents a significant difference from the equivalent concentration of curcumin alone (p < 0.05). (b) Western blots of cell cycle/apoptosis-related proteins in the 3 colon cell lines following a 24 or 8 hr treatment with curcumin +/− oxaliplatin (n = 3).

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HT29 cells responded similarly to HCEC following 24 hr treatments. Oxaliplatin resulted in a significant S-phase accumulation (71% of cells in total), but there was little significant effect on any phase of the cell cycle with curcumin, although when combined, curcumin did abrogate the oxaliplatin-induced S-phase arrest. At 48 and 72 hr, the oxaliplatin-induced S-phase arrest was no longer significant. Instead, significant G2 arrest was observed when oxaliplatin was combined with 5 μM curcumin. Again, this was abrogated in combination with higher doses of curcumin.

Five μM oxaliplatin resulted in 54% of the HCT116 cells arresting in G2/M, while 5 μM curcumin resulted in 37%, which rose to 63 and 65% for 10 and 20 μM curcumin respectively. The combination of oxaliplatin with curcumin for 24 hr enhanced the G2/M arrest further still at 10 μM, but this was reversed at 20 μM. Following a 48 hr treatment, oxaliplatin-induced G2/M arrest reached 70%, while both 10 and 20 μM curcumin caused ∼80% of cells to arrest in G2/M. This arrest was marginally enhanced when oxaliplatin was combined with the lower doses of curcumin but abrogated at 20 μM. At 72 hr oxaliplatin treatment, virtually all of the cells were in G2/M, whilst this effect of curcumin alone was lost, suggesting that oxaliplatin-mediated arrest was irreversible in this cell line, whilst curcumin-mediated arrest was not. Almost complete G2/M arrest was maintained with oxaliplatin + 5 μM curcumin, but combination of oxaliplatin with 20 μM curcumin released ∼30% of cells into S phase.

At an earlier time point of 8 hr, no significant arrest was detected with either treatment separately in the HCT116 cells. Significant S-phase accumulation was observed, however, with the combination (10 and 20 μM curcumin) compared with the DMSO control or oxaliplatin alone (data not shown).

Effect of combined treatments on expression of proteins related to cell cycle

Further investigation into the effects of these treatments on cell cycle and survival-related proteins was carried out following a 24 hr treatment (Fig. 5b). Oxaliplatin resulted in a modest increase in p21 levels in the HCEC cells. Curcumin elevated p21 at 5 μM and p53 levels at 20 μM. Following combined treatments, p21, p53 and cdc2 were elevated compared with the control, and similar to oxaliplatin alone. There was no significant alteration to survivin expression following any treatment.

Expression patterns in the HT29 cells remained largely unchanged following curcumin or oxaliplatin treatments. Levels of mutant p53 did not alter and p21 was not re-expressed (data not shown). Oxaliplatin alone significantly decreased cdc2 (62% of DMSO control ± 14%) and survivin levels in this cell line, whilst the co-treatment resulted in increased phospho-cdc2. No significant changes to these proteins were observed at the earlier time point of 8 hr (data not shown).

The most obvious changes were observed in the HCT116 cells. Oxaliplatin treatment resulted in an approximately 20-fold induction of p21, whereas curcumin resulted in a 2- to 3-fold induction. The combined treatment (10 and 20 μM curcumin) decreased oxaliplatin-induced levels of p21 by 30–40%. p53 levels were increased approximately 8-fold by a 5 μM oxaliplatin treatment and about 4-fold with curcumin. The combinations resulted in a greater than additive increase in p53 expression to levels of 16-fold above that of the control. Chk1 levels were decreased in the presence of oxaliplatin but not by curcumin alone, whereas phospho-chk1 levels remained unchanged following any treatment. Cdc25c levels were affected similarly to chk1. Phospho-cdc2 levels were significantly decreased by oxaliplatin but were elevated in the presence of curcumin alone. Survivin was upregulated by 5 μM curcumin, but decreased by oxaliplatin alone and by the combinations. At 8 hr, the only proteins significantly affected by the treatments were p21 and p53. p21 was induced 6-fold by oxaliplatin alone and up to 4-fold by curcumin alone, with the combination resulting in a 23-fold induction (20 μM curcumin). Induction of p53 was less marked than for 24 hr, with a 2-fold induction observed with the combined treatment.

Effect of combined treatments on cell proliferation in HCT116 p53−/− and p21−/− cell lines

Oxaliplatin alone inhibited proliferation in the knockout cell lines to a similar magnitude observed for HT29 and HCT116 wt cell lines (Fig. 6). However, there was a marked difference when treated with curcumin alone and with the combination treatments. Curcumin did not inhibit growth to any extent in either of the knockout cell lines at concentrations up to 20 μM (144 hr). Following a combined treatment of 5 μM oxaliplatin with 1 μM curcumin, proliferation was significantly inhibited in both p53−/− and p21−/− cell lines, but this was by 61- and 52-fold respectively (a similar magnitude to the HT29 cells), compared with their wt counterparts, which exhibited a decrease of 344-fold. However, at higher combined doses, inhibition of proliferation exhibited a similar fold-decrease to the wild type cells.

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Figure 6. Mean fold change in cell number (n = 4) from DMSO control in HCT116 p53−/− and HCT116 p21−/− cells following treatment with curcumin +/− oxaliplatin at 144 hr +/-SEM. * represents significant difference from DMSO control, “a” represents a significant difference from oxaliplatin alone, and “b” represents a significant difference from the equivalent concentration of curcumin alone (p < 0.05).

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Discussion

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

The induction of apoptosis by oxaliplatin in the HCT116 and HT29 cell lines (Fig. 1) was consistent with previous studies,15, 16, 19 as was the lack of sensitivity in the normal-derived HCEC cell line.20 While the difference in sensitivity between HCT116 and HT29 was minimal, significant differences in expression of cell cycle related proteins were observed. Wild type p53 was induced in HCT116 cells, while no change was apparent in HT29 cells with mutant protein.

The necessity for p53 in oxaliplatin-induced apoptosis remains ambiguous. Hayward et al.,15 showed p53-null HCT116 cells to have a greater ability than wild type cells to recover following oxaliplatin treatments, primarily due to less induction of apoptosis. Conversely, Gourdier et al.,21 concluded that the p53 pathway has only a minor role in oxaliplatin-induced apoptosis, is probably not involved in oxaliplatin resistance,21 and is not a suitable marker for predicting response to oxaliplatin16. From our study, it is likely that survival signaling in response to oxaliplatin exhibits cell line specificity; HCT116 cells require p53 for the oxaliplatin/curcumin-induced apoptotic response, whereas mutated p53 in HT29 cells does not prevent this chemotherapy-induced apoptosis.

P21 was induced in HCEC and HCT116 cells. Bunz et al.,22 postulated a requirement for both p53 and p21 to sustain G2 arrest following DNA damage during which, damage can be repaired, or pro-apoptotic proteins upregulated to eliminate those cells which cannot be repaired. Oxaliplatin also affects other damage-responsive proteins involved in cell cycle and cell death, including cdc2 and survivin. Both are intimately linked to the G2/M phase of the cell cycle and can be regulated in both a p53-dependent or independent fashion. During G2, cdc2 is complexed with cyclin B and is kept inactive by phosphorylation on threonine 14 and tyrosine 15. Both of these residues are dephosphorylated by cdc25 to enable the onset of mitosis.23 This can be regulated by p53-dependent transcription of p21, which can directly inhibit cdc2, or in a p53 independent manner by DNA-damage-stimulated activation of ATM/ATR, which activates Chk1/2 causing 14-3-3 binding of cdc25. This prevents cdc25 from activating (dephosphorylating) cdc2 and thus prevents the onset of mitosis.24 Survivin is a member of the inhibitor of apoptosis (IAP) family, which is selectively expressed at G2/M, where it localizes to mitotic spindle microtubules,25 and is phosphorylated and stabilized by cdc2 at the G2 transition phase.

Rather than exhibiting an increase in phosphorylated inactive cdc2, HCT116 cells (48 hr) showed downregulation of total protein, with consequent decrease in phospho-cdc2 and survivin. Fujie et al.,26 described a G2/M arrest of greater than 50% following a 48 hr oxaliplatin treatment in the HT29 cells, supported by a decrease in both cdc2 and survivin. However, little effect on cell cycle was observed in the present study in HT29 cells, despite similar decreases in these proteins at this time point.

While both agents were antiproliferative in their own right and particularly so in combination, there were distinct differences in sensitivity between cell lines. The normal-derived HCEC cells were least sensitive to both curcumin and oxaliplatin, whereas HT29 cells were sensitive to curcumin concentrations as low as 0.1 μM (data not shown). HCT116 cells were less sensitive to curcumin, but more sensitive to oxaliplatin than the HT29 cells. This would suggest that the compounds either target differing antiproliferative pathways or that regulation of the primary target differs between the two tumor cell lines, which may be a function of p53 status. The decreased sensitivity observed in the HCT116 knockout cell lines gives further evidence that both p53 and p21 play an important role in governing the response of this cell line to therapeutic agents, and it was of particular interest that wt p53 and p21 were essential for curcumin sensitivity. However, when curcumin and oxaliplatin were combined, there was still a significant fold-decrease in cell number when compared with oxaliplatin alone. The minimal effect of curcumin and oxaliplatin on HCEC cells both singly and in combination, reinforces their potential in clinical regimes with regards to preventing unwanted toxicities.

Assessment of cell cycle in response to treatments revealed that HCT116 cells underwent significant G2/M arrest in response to either compound. An enhanced, almost complete, arrest was observed when the two were in combination, but ∼30% of cells were released from this arrest at the highest dose of curcumin.

Interestingly, p53 protein expression was increased 16-fold by the combination of oxaliplatin with curcumin in the HCT116 cells (24 hr), whilst oxaliplatin-induced p21 expression was reduced following combination with 10 and 20 μM curcumin, although greatly enhanced induction of p21 following combined treatments is apparent at the earlier time point of 8 hr. These concurrent events may account for the less pronounced G2/M arrest at the high dose of curcumin. Curcumin-induced arrest appeared reversible, whereas oxaliplatin-induced G2/M arrest was not. This further suggests that mechanisms differ between compounds. Oxaliplatin is primarily a DNA-damaging agent that makes for inefficient repair due to formation of bulky DNA adducts. In p53wt cells, upregulation of p53 in conjunction with initiation of the DNA damage checkpoint response likely contributes significantly to the ability of oxaliplatin to initiate both cell cycle arrest and the apoptotic response. Evidence for curcumin as a DNA-damaging agent however, remains ambiguous, with some reports that it may cause oxidative damage at low micromolar doses. The results presented here suggest curcumin mediates its effects mainly via induction of apoptosis, as cell cycle inhibition appears reversible. Further investigation as to whether it activates DNA damage-regulated checkpoints is ongoing, although preliminary evidence suggests that both chk1 and cdc25c at least, remain unaffected by curcumin but are downregulated in the presence of oxaliplatin in the HCT116 cells at 24 hr.

No oxaliplatin-induced G2/M arrest was observed in the HT29 cells at any time point. Cells possessing non-functional p53 have been observed to continue through to mitosis following a brief arrest, where it is likely they undergo mitotic catastrophe during metaphase27, 28 due to their inability to repair gross chromosomal abnormalities. Both curcumin (20 μM) and oxaliplatin served to prolong the S-phase at 24 hr, which would result in a net slowing of cell cycling, possibly indicating activation of a p21/p53-independent checkpoint mechanism. It has been shown that benzo(a)pyrene dihydrodiol epoxide (BPDE) causes chk1-regulated S-phase arrest in p53-deficient lung cancer cells.29 Similarly, the chemopreventive agent resveratrol enhanced S-phase arrest in ovarian carcinoma cells due to cdc2 (tyr 15) phosphorylation via chk1/2.29 A non-significant increase in cdc2 phosphorylation was observed in the HT29 cells particularly in combination with curcumin, which may predict that these cells are similarly regulated by p53/p21 -independent checkpoint mechanisms.

No induction of caspase activity was observed in the HCEC cells, which again reflected their relative insensitivity to either agent. Caspase 8 and 9 activity was induced by both oxaliplatin and curcumin in the tumor cell lines, with higher levels of caspase 8 cleavage (particularly in the HCT116 combination treatments) suggesting this to be the apical caspase, although concurrent caspase 9 induction indicates apoptosis to be mitochondrial-mediated.30 In HCT116 cells, induction of caspase 3/7 activity by 5 μM oxaliplatin and 5 μM curcumin in conjunction with the cell cycle arrest (24 hr) reveals why proliferation was reduced by up to 3-fold in this cell line following a 24 hr treatment. Induction of caspase activity was enhanced in both the tumor cell lines when the 2 agents were combined, although such enhancement was less obvious in the annexin V binding assay. This would suggest that whilst increased caspase activation may be sufficient to explain the decreased proliferation following combined treatment in the HT29 cells, it cannot wholly account for the much greater fold-decrease observed following the combination in HCT116 cells. This is likely to be due to the combined affect of G2/M arrest and induction of apoptosis. Further investigations are required to reveal whether the potent oxaliplatin-induced G2/M arrest and its potentiation by low-dose curcumin in the HCT116 cells is a necessary event for induction of apoptosis, or whether these 2 antiproliferative mechanisms act independently from one another.

There is growing evidence for enhanced cytotoxicity by combining targeted drugs such as the EGFR inhibitor ZD-1839 (Iressa) with oxaliplatin,19, 31, 32 alluding to synergistic action independent from the additive effects of each agent. Recently, the COX-2 inhibitors etodolac and celecoxib were found to enhance oxaliplatin-induced cytotoxicity in the RKO colorectal cancer cell line, possibly due to associated inhibition of survivin.33 Such findings are relevant for curcumin in combination with oxaliplatin, since we and others have shown this dietary agent to inhibit both EGFR-13, 34 and COX-2-mediated signaling in tumor-derived cell lines,35, 36 with further potential for synergism through its well-documented ability to inhibit NF-kB signaling.37, 38 Of primary importance is the lack of systemic toxicity observed so far in phase I clinical trials with very high doses of curcumin, which is in contrast to many therapeutic drugs.

This study is the first to show that the diet-derived polyphenol curcumin is able to achieve similar indices of efficacy in vitro, to the chemotherapeutic agent oxaliplatin at therapeutically achievable concentrations in both p53 mutant and wild type colorectal carcinoma cell lines. Both compounds selectively target tumor cells, and some therapeutic indices are markedly enhanced when the agents are combined. There is great potential for curcumin to significantly contribute to therapeutic strategies, particularly when in combination with traditional therapeutic agents. Further work is now ongoing to expedite its use within clinical regimes.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

We thank Prof. G. Cohen (MRC Toxicology Unit, Leicester, UK) for provision of the caspase 8 antibody, and Prof. B. Vogelstein (John Hopkins University, Baltimore, MD, USA) for kind provision of HCT116 p21−/− and p53−/− cell lines.

References

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
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