Cancer Letters

Cancer Letters

Volume 224, Issue 1, 16 June 2005, Pages 53-65
Cancer Letters

Antitumor effects of curcumin, alone or in combination with cisplatin or doxorubicin, on human hepatic cancer cells. Analysis of their possible relationship to changes in NF-kB activation levels and in IAP gene expression

https://doi.org/10.1016/j.canlet.2004.10.051Get rights and content

Abstract

The hepatic cancer HA22T/VGH cell line, which constitutively expresses activated nuclear factor-kappaB (NF-kB), was chosen as a model to examine the antitumor activity of curcumin, also in relationship to its possible influences on the activation of the transcription factor and on the expression of the inhibitory of apoptosis proteins (IAPs) and of other NF-kB target genes. Curcumin exerted cell growth inhibitory and apoptotic effects, related, at least part, to free radical generation and mainly dependent on caspase-9 and -3 activation. The combination of curcumin with cisplatin resulted in a synergistic antitumor activity and that with doxorubicin in additivity or sub-additivity. Curcumin exerted biphasic changes in the levels of NF-kB, with an increase at 8 h after its administration and a decrease at 16 h. For the combinations of curcumin with the other drugs, the levels of the transcription factor were lower than those predicted from the effects of the single agents, especially with a blunting of the remarkable increases in NF-kB activation induced by doxorubicin. Except for Bcl-2, the HA22T/VGH cells expressed different other genes, including the IAPs, implicated in cell proliferation and survival. Curcumin determined early changes in COX-2 and c-myc mRNAs, which were down-regulated, and in livin mRNA, which was up-regulated. Later it decreased Bcl-XL mRNA and increased Bcl-XS and c-IAP-2 mRNAs. Cisplatin and doxorubicin exerted distinct effects on gene expression. The cytotoxic interactions between curcumin and these agents were accompanied by synergistic (in particular with cisplatin) or additive effects of decrease in the expression of different genes, including c-myc, Bcl-XL, c-IAP-2, NAIP and XIAP. However, the combinations attenuated also certain other influences on mRNA expression of the single agents, like, for example, the increases in Bcl-Xs given by curcumin and doxorubicin. Overall, the effects of the drugs, alone or in combination, on tumor cell growth, cell death and gene expression did not show a simple relationship to the relative influences on NF-kB activation, inferring that they can be due also to other mechanisms.

Introduction

There is an increasing evidence that the inability of the cells to undergo apoptosis may critically contribute to the genesis and progression of cancer and represent an important cause of tumor drug resistance. This well applies to hepatocellular carcinoma (HCC), a highly aggressive tumor which responds poorly to currently available therapies [1]. Indeed, this cancer frequently shows the over-expression of anti-apoptotic factors like the Bcl-2 family members and the IAPs (inhibitory of apoptosis proteins) [2], [3], [4], [5], [6]. IAPs, which in humans include c-IAP-1, c-IAP-2, XIAP, NAIP survivin and livin-α (known also as ML-IAP), have shown a remarkable ability of blocking cell death induced by very many non-related triggers [7], [8], [9]. They can in fact bind and potently inhibit key effector caspases. Further, some of them, like XIAP, can trigger the proteasome degradation of pro-apoptotic substrates, like caspases and Smac [7], [10], [11].

The expression of XIAP and other IAPs can be up-regulated by nuclear factor-kappaB (NF-kB) [12], [13], which, in turn, is frequently constitutively activated in HCC [14], [15]. Thus, the inhibition of the transcription factor might be of help to antagonize the IAPs as well as other NF-kB target genes (e.g. COX-2, Bcl-XL and c-myc) involved in the adverse biology of this cancer [3], [16], [17], [18], [19]. To address this aspect, we have examined the effects of curcumin, alone or in combination with conventional anticancer agents like cisplatin and doxorubicin, on the HCC cell line HA22T/VGH. Curcumin, a polyphenolic compound extracted from rhizomes of Curcuma species, has in fact been shown to possess interesting anti inflammatory and antitumor properties, which, at least in part, appear be linked to its ability to suppress the activation of NF-kB [20], [21], [22]. Since the HA22T/VGH cell line constitutively expresses activated NF-kB, we thought that it might be an appropriate model to examine the possible relationship of the antitumor activities of curcumin to its influences on the activation of NF-kB as well as on the expression of IAPs and other NF-kB target genes.

Section snippets

Agents

Curcumin, cisplatin and N-acetyl-l-cysteine (NAC) were purchased from Sigma-Aldrich Srl, Milan, Italy, doxorubicin from Pharmacia, Milan, Italy, z-Asp(OCH3)-Glu(OCH3)-Val-Asp(OCH3)-fmk (z-DEVD-fmk) from Calbiochem, Milan, Italy and z-Ile-Glu-Thr-Asp-fmk (z-IETD-fmk) and z-Leu-Glu (OMe)-His-Asp (OMe)-fmk (z-LEDH-fmk) from Alexis Biochemicals, Laufelfingen, Switzerland.

Cell culture

HA22T/VGH is a poorly differentiated hepatoma cell line which contains HBV integrants. It was kindly provided by Professor M.

Antitumor and cell death effects of curcumin alone or in combination with cisplatin or doxorubicin

The antitumor effects of curcumin on HA22T/VGH cells were first evaluated by MTS assay: after 72 h of treatment the concentrations of the drug which caused 50 and 70% inhibition of cell growth were 39.1 and 79.2 μM, respectively. We determined also BrdU incorporation into DNA and this assay was more sensitive, since the concentrations of curcumin which inhibited of 50 and 70% DNA synthesis after 48 h of treatment were 9.8 and 12.3 μM, respectively. Curcumin induced apoptosis, which was evaluated by

Discussion

Our current research interests deal with possible strategies able to overcome the resistance to drugs and apoptosis, possibly related to IAP expression, which characterizes a cancer with a so poor prognosis such as HCC. Inhibition of NF-kB might be of help to antagonize the IAPs and other NF-kB target genes in this tumor and in this context we have focused on curcumin. This interesting polyphenolic compound is endowed with different mechanisms which may explain its anti-inflammatory and

Acknowledgements

This work was supported by Progetto Strategico Oncologia ‘Terapia preclinica molecolare in oncologia’ MIUR-CNR.

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