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Introduction

At the earliest time point when apoptosis was first detected images are highlighted arrows. Mitochondrial changes arrowheads are shown. The results indicate that a very low quantity of 5-FU 0. Therefore, we determined the behavior of these two CRC cells in the various phases of the cell cycle by flow cytometric analysis Fig.

Treated cells were harvested and processed for flow cytometric analysis. The most significant effect in both single and combined treatment was a time- and concentration-dependent reduction of cells in the G1-phase and accumulation of cells in the S-phase of the cell cycle. Besides the changes in G1- and S-phase distribution, the number of cells undergoing apoptosis was markedly elevated with treatment. Interestingly, the effects of the combination treatment clearly exceeded those of the single treatments.

Cell cycle analysis was performed by flow cytometry. These studies were performed in triplicate and the results presented are mean value with standard deviations from three independent experiments. Activation of caspases induces apoptosis in cells. Furthermore, activation of caspase-8 extrinsic pathway and -9 intrinsic pathway stimulates caspase-3, which in turn induces PARP cleavage [34].

It is important to note that these effects were more pronounced in combination treatment with curcumin and 5-FU than in single treatments. Whole cell lysates were prepared and analyzed by western blotting for A: expression or cleavage of pro-apoptotic proteins caspase-8, caspase-9, caspase-3, PARP and Bax, and of anti-apoptotic protein BCL-xL B: expression of cyclin D1. This result suggests that pretreatment with curcumin sensitizes the cells to 5-FU-induced cyclin D1 inactivation, most likely through the suppression of cell proliferation proteins.

Activation of caspase-8 induces cytochrome c release from mitochondria, which then activates caspase-9 and -3 [34].

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Cells were treated with 5-FU or curcumin alone or pretreated with curcumin and then treated with 5-FU as described above. The cytosolic cytochrome c increased in the presence of 5-FU or curcumin and even more in the combination treatment with both agents. At the same time, there was a corresponding decrease in the mitochondrial compartment. Thus, these results suggest that pretreatment with curcumin sensitizes the cells to 5-FU-induced apoptosis Fig.

Mitochondrial and cytoplasmic cell fractions were prepared and analyzed by western blotting using antibodies against cytochrome c. Taken together, these results support a functional role for the PI-3kinase pathway in 5-FU-induced signaling pathway. HCT Fig. The results from the immune complex kinase assay, performed as described in Materials and Methods, showed that 5-FU induced the activation of IKK in a time-dependent manner, while pretreatment of cells with curcumin or PI-3K inhibitor wortmannin followed by stimulation with 5-FU blocked the 5-FU-induced effects on the activation of IKK to a similar extent Fig.

Cells were lysed and immune complex kinase assays were performed as described in Materials and Methods. The incidence of CRC and its mortality rate are steadily increasing. Currently available chemotherapeutic agents for the treatment of CRC are associated with numerous side effects, development of treatment resistance and, until now, have not improved patient survival.

Therefore, non-toxic and more selective pharmacotherapies are needed for CRC. Combination treatments may enhance the responses of CRC cells to chemotherapeutic agents.

1. Introduction

Epidemiological studies suggest that plant polyphenols have the potential to sensitize tumor cells to chemotherapy by specifically suppressing signaling pathways that are involved in the development of treatment resistance induced by conventional chemotherapeutic drugs.

How these polyphenols protect and display proliferative effects in healthy cells while sensitizing tumor cells and making them more susceptible to chemotherapeutics is not fully understood. Several studies have shown that the phytochemical curcumin displays anti-tumor effects in various solid tumors and potentiates the effect of a number of chemotherapeutic agents leading to increased sensitivity even in drug-resistant cancer cells [25] , [39] , [40].

The chemotherapeutic agent 5-FU is routinely used as a standard therapy for treatment of CRC, but with limited success [41]. In this study we hypothesized that curcumin might potentiate the anti-proliferative effect of chemotherapeutic agents in advanced CRC and inhibit metastasis. Therefore the primary goal of this study was to determine whether curcumin can sensitize CRC cells to 5-FU and to investigate the mechanism of action of this chemosensitization.

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This study demonstrates that combining curcumin and 5-FU leads to a significant suppression of growth and viability of CRC cells, indicating that curcumin sensitizes the 5-FU surviving CRC cells to treatment. Therefore, there might be a mechanistic relationship between the combined drug action and DNA damage repair pathway. The deficiency of the mismatch repair MMR system in tumor cells could be important for clinical resistance to chemotherapy and this should be carefully considered for the selection and use of the therapeutic agents. This is consistent with the observations of Sargent et al.

The S phase of the cell cycle is connected with the major cellular event of replication [44]. The combination of curcumin and 5-FU caused increased apoptosis in CRC cells compared to treatment with the single agents.


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The data presented suggest that HCT cells exhibit a higher susceptibility to the combination therapy than to single agents, suggesting that this treatment may be an effective therapeutic strategy for targeting chemoresistant cancer cells. Moreover, the combination of phytochemicals with standard chemotherapy can significantly reduce the dosage of the chemotherapeutic agents and thereby minimize adverse side effects and drug toxicity for the patients.

Curcumin is a well-tolerated phytochemical, which could prevent primary tumor formation or tumor recurrence. Here, we provide evidence that curcumin may have chemopreventive potential against CRC by affecting multiple cell signaling molecules. The release of mitochondrial mediators of apoptosis such as cytochrome c and apoptosis inducing factor is associated with mitochondrial damage.

Consistent with its function in the PI-3K signaling cascade, the inhibition of PI-3K by curcumin and subsequent blockage of Akt may induce apoptosis.

The tyrosine kinase Src, that regulates cellular signaling pathways, has been linked with different stages of tumor progression such as CRC metastasis [53] , [54] and implicated in treatment resistance to chemotherapeutic agents [55]. Indeed, it has been reported that suppression of Src signaling sensitizes tumor cells to chemotherapies [56] , [57]. In agreement with these findings, we clearly demonstrate that curcumin alone or in combination with 5-FU markedly decreases Src expression in a dose-dependent manner in HCT cells, suggesting the inhibition of Src activity by curcumin imparts higher sensitivity to 5-FU.

Overall, our results indicate that the combination of curcumin, which is a pharmacologically safe natural compound, with conventional chemotherapeutics like 5-FU could provide an improved strategy for colon cancer therapy. The authors gratefully acknowledge the excellent technical assistance provided by Ms. Ursula Schwikowski.

National Center for Biotechnology Information , U.


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PLoS One. Published online Feb Bharat B. Aggarwal, Editor. Author information Article notes Copyright and License information Disclaimer. The University of Texas M. Competing Interests: The authors have declared that no competing interests exist. Received Jul 9; Accepted Jan This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.

This article has been cited by other articles in PMC. Abstract Objective Development of treatment resistance and adverse toxicity associated with classical chemotherapeutic agents highlights the need for safer and effective therapeutic approaches. Conclusions Combining curcumin with conventional chemotherapeutic agents such as 5-FU could provide more effective treatment strategies against chemoresistant colon cancer cells. Introduction Colorectal cancer CRC is one of the leading causes of death in men and women, and ranks among the third most common cancers globally [1].

Quantification of apoptotic cell death Ultrathin sections of the samples were prepared and evaluated with an electron microscope TEM 10; Zeiss. Isolation of mitochondrial extracts for detection of cytochrome c-release Cells were washed in 1 ml ice cold PBS and placed in ice-cold mitochondria isolation buffer 0.

Immune complex kinase assay Immune complex kinase assay was performed as previously described in detail [33].

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Results This study was designed to investigate how curcumin inhibits the proliferation of CRC cells and potentiates the effects of the chemotherapeutic agent 5-FU in an in vitro model of human CRC cells. Open in a separate window. Figure 1.

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Figure 2. Figure 3. Figure 4. Figure 5. Figure 6. Figure 7. Figure 8. Figure 9. Discussion The incidence of CRC and its mortality rate are steadily increasing. Figure Acknowledgments The authors gratefully acknowledge the excellent technical assistance provided by Ms. Funding Statement The authors have no support or funding to report. References 1. Cancer : — CA Cancer J Clin 59 : — Benson AB 3rd New approaches to the adjuvant therapy of colon cancer.

Oncologist 11 : — Ohtsu A Chemotherapy for metastatic gastric cancer: past, present, and future. J Gastroenterol 43 : — Biochem Pharmacol 64 : — Aggarwal BB Nuclear factor-kappaB: the enemy within. Cancer Cell 6 : — Science : — Nat Med 5 : — Ann N Y Acad Sci : — Cell 87 : 13— Oncogene 18 : — Cell : 57— Cell Cycle 8 : — Berdasco M, Esteller M Aberrant epigenetic landscape in cancer: how cellular identity goes awry. There are treatments available for metastatic breast cancer.

The ACS explains that current treatment options are "very unlikely to cure metastatic breast cancer," but there are some therapies that can shrink tumors, slow their growth, and improve symptoms, helping patients live longer. Typically, these cancers are treated with systemic, or full-body, approaches , including chemotherapy , hormone therapy since some breast cancers interact with hormones , and drugs that specifically target the growth and spread of cancer cells, according to the ACS.

It's not clear exactly clear whether Newton-John is currently using any systemic treatments like these — "Sunday Night" only reported that she has undergone radiation and that she has switched to a "healthier diet. There are moments. I'm human But my husband is always there and he's always there to support me. I believe I will win over it. Caroline Praderio. Facebook Icon The letter F.

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Link icon An image of a chain link. It symobilizes a website link url. Email icon An envelope. It indicates the ability to send an email. Fliboard icon A stylized letter F. Twitter icon A stylized bird with an open mouth, tweeting. Pinterest icon The letter "P" styled to look like a thumbtack pin. Olivia Newton-John is facing a new cancer diagnosis. Alberto E. She first found a lump in her breast in