L-carnosine dipeptide overcomes acquired resistance to 5-fluorouracil in HT29 human colon cancer cells via downregulation of HIF1-alpha and induction of apoptosis.

a*Iovine B, aGuardia F, bIrace C, aBevilacqua MA.

aDipartimento di Medicina Molecolare e Biotecnologie Mediche, Università degli Studi di Napoli Federico II, via Sergio Pansini 5, 80131 Napoli, Italy

bDipartimento di Farmacia, Università degli Studi di Napoli Federico II, via Domenico Montesano 49, 80131 Napoli, Italy

* Corresponding author. E-mail addresses: barbara.iovine@unina.it, iovine@dbbm.unina.it



Hypoxia-inducible factor (HIF-1α) protein is over-expressed in many human cancers and is a major cause of resistance to drugs. HIF-1α up-regulation decreases the effectiveness of several anticancer agents, including 5-fluorouracil (5-FU), because it induces the expression of drug efflux transporters, alters DNA repair mechanisms and modifies the balance between pro- and antiapoptotic factors. These findings suggest that inhibition of HIF-1α activity may sensitize cancer cells to cytotoxic drugs. We previously reported that L-carnosine reduces HIF-1α expression by inhibiting the proliferation of colon cancer cells. In the present study we investigated the effect of L-carnosine on HT29 colon cancer cells with acquired resistance to 5-FU. We found that L-carnosine reduces colon cancer cell viability, decreases HIF-1α and multi-drug resistant protein MDR1-pg expression, and induces apoptosis. Moreover, the L-carnosine/5-FU combination lowers the expression of some chemoresistance markers. The combination index evaluated in vitro on the HT29-5FU cell line by median drug effect analysis reveals a significant synergistic effect.

KEYWORDS: 5-FluorouracilApoptosis; Chemoresistance; Hypoxia-inducible factor-1; Multi-drug resistancel-carnosine

PMID: 27234614



L-carnosine (β-Ala-His) is a naturally occurring histidine dipeptide, normally found in brain, kidney and in large amounts in muscle (1). L-carnosine is known to possess numerous functions, including antioxidant activity, ability to chelate metal ions, as well as anti-inflammatory and anti-senescence properties (2). In the last few years the attention of several researchers has been focused on the capacity of L-carnosine to inhibit cell proliferation in many various human tumor cell lines in vitro and in vivo mouse models. We have previous demonstrated that L-carnosine decreases cell proliferation in colon cancer cell line, by affecting ATP and ROS production. Furthermore, we found that the anti-proliferative effect correlates with a decrease in the expression of the Hypoxia Inducible Factor 1 alpha (HIF-1α), a central regulator of the adaptation responses of cancer cells to hypoxia (3-4-5). HIF-1α is an O-regulated subunit, that links a constitutively expressed HIF-1β subunit to form the heterodimeric transcription factor HIF-1. The HIF-1 complex recognizes and binds to the hypoxia responsive element (HRE) inducing the expression of genes involved in angiogenesis, iron metabolism, in glucose metabolism modulation, cell proliferation, survival, invasion and resistance to chemotherapy (6). The hypoxic microenvironment, common feature of tumor cells, and particularly the HIF-1α expression are the mainly responsible for the multi-drug resistance (MDR). Indeed, HIF-1α influences the expression of a multidrug efflux transporter (MDR1/P-gp), represses apoptosis, affecting the Bax and Bcl-2 expression and seems to be a principal cause for the resistance to 5-fluorouracil, the first chemotherapeutic agent used to treat colon cancer (7). These findings suggest that the inhibition of HIF-1α activity may sensitize cancer cells to 5-FU.

In this study we focus the attention on the study of the effect of L-carnosine treatment on HT29 colon cancer cells selected by adaptation to 5- fluorouracil (HT29-5FU).

We find that in the 5-FU resistant HT-29 cell line (HT29-5FU) mRNA and HIF-1α protein expression were higher compared to the parental wild-type 5-FU sensitive HT29 cells, in which the Western blot analysis shows an HIF-1α signal barely detectable. The expression of HIF-1α could explain the evident resistance to 5-FU of the selected cell line. In HT29-5FU cells we also find higher mRNA and protein expression of the anti-apoptotic gene Bcl-2, and lower mRNA and protein expression of the pro-apoptotic gene Bax with respect to HT29 cells. L-carnosine treatment decreases HIF-1α expression and influences the Bax and Bcl-2 expression, inducing apoptosis. In addition, the clonogenic assay has demonstrated that L-carnosine reduces the ability to form colonies and has cytotoxic effects on the cells. The treatment shows also a reduction in P-gp protein expression, which has been directly linked to the regulation of apoptosis mediated by Bcl-2, Bax and caspases. We thereby suppose that the apoptotic effect of L-carnosine treatment on HT29 5FU-resistant cells depends on the high expression of HIF-1α. Indeed, we observe no effect on HT29 wild-type treated with L-carnosine. This analysis let us to hypothesize that L-carnosine overcomes acquired resistance to 5-FU, by reducing HIF-1α expression (Figure1).

The most interesting aspect of our research is that the combination index, calculated by the Chou-Talalay equation, shows a significant synergistic interaction between L-carnosine and 5-FU. Indeed, L-carnosine in combination with 5-FU reduces cell viability and concurrently increases the number of apoptotic cells with respect to the treatment with a single drug. In addition, the combination treatment reduces the expression of some markers of resistance to 5-FU such as the HSP27 protein and the autophagy, which represent one of the major mechanisms by which cells develop resistance to chemotherapy drugs (9-10).

The importance of this study: The resistance to drugs as well as the drug cellular uptake represent the main obstacles in the development of an effective cancer therapy. Indeed, the new anticancer strategies aim to selectively deliver drugs towards cancer cells, thus decreasing both the no specific uptake into normal cells and the chemoresistance. L-carnosine is a natural dipeptide endogenously synthesized, bioavailable and absorbed primarily via intestinal epithelium cells. Moreover, these features and the ability to induce apoptosis in colon cancer cells 5FU-resistant, suggest that L-carnosine may be a good adjuvant candidate for colon cancer treatment in combination with 5-FU.


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