Br J Pharmacol. 2013 Sep;170(2):233-44. doi: 10.1111/bph.12255.

Solid lipid nanoparticles of cholesteryl butyrate inhibit the proliferation of cancer cells in vitro and in vivo models.

Minelli R, Occhipinti S, Gigliotti CL, Barrera G, Gasco P, Conti L, Chiocchetti A, Zara GP, Fantozzi R, Giovarelli M, Dianzani U, Dianzani C.

Department of Drug Science and Technology, University of Turin, Torino, Italy.

 

ABSTRACT

BACKGROUND AND PURPOSE: Solid lipid nanoparticles containing cholesteryl butyrate (cholbut SLN) can be a delivery system for the anti-cancer drug butyrate. These nanoparticles inhibit adhesion of polymorphonuclear and tumour cells to endothelial cells and migration of tumour cells, suggesting that they may act as anti-inflammatory and anti-tumour agents. Here we have evaluated the effects of cholbut SLN on tumour cell growth using in vitro and in vivo models.

EXPERIMENTAL APPROACH: Cholbut SLNs were incubated with cultures of four tumour cell lines, and cell growth was analysed by assessing viability, clonogenic capacity and cell cycle. Effects on intracellular signalling was assessed by Western blot analysis of Akt expression. The in vivo anti-tumour activity was measured in two models of PC-3 cell xenografts in SCID/Beige mice.

KEY RESULTS: Cholbut SLN inhibited tumour cell line viability, clonogenic activity, Akt phosphorylation and cell cycle progression. In mice injected i.v. with PC3-Luc cells and treated with cholbut SLN, . in vivo optical imaging and histological analysis showed no metastases in the lungs of the treated mice. In another set of mice injected s.c. with PC-3 cells and treated with cholbut SLN when the tumour diameter reached 2 mm, analysis of the tumour dimensions showed that treatment with cholbut SLN substantially delayed tumour growth.

CONCLUSION AND IMPLICATIONS: Cholbut SLN were effective in inhibiting tumour growth in vitro and in vivo. These effects may involve, in part, inhibition of Akt phosphorylation, which adds another mechanism to the activity of this multipotent drug.

© 2013 The British Pharmacological Society.

KEYWORDS: cholesteryl butyrate, clonogenic assay, solid lipid nanoparticles, tumour growth in vivo

PMID: 23713413

 

SUPPLEMENT:

In an earlier work, we showed that cholbut SLN may have an effect in tumor cell dissemination because it inhibited adhesion of tumor cell lines to vascular endothelial cells and their migratory activity, which suggested that cholbut SLN may work as an anti-metastatic compound (Minelli et al., 2012). The present work further strengthens findings on the anti-tumor effectiveness of cholbut SLN by showing its ability to inhibit cancer cell growth both in vitro and in vivo.

Our data show that cholbut SLN inhibited tumor cell growth in vitro at higher doses than those effective on tumor cell adhesion and migration. The effect was dose- and time-dependent and clearly more potent than that displayed by NaBut, which has been found to be ineffective on tumor cell adhesion and migration in our previous work. Moreover, NaBut is selectively effective in some tumor cell lines, whereas cholbut SLN were active on all cell lines tested. Wilson et al., 2010 demonstrated that cancer cell sensitivity to NaBut correlated with its capacity to inhibit HDAC activity. The cholbut SLN effect would be independent from HDAC inhibition because our previous work showed that these cholbut SLN doses do not inhibit HDAC (Minelli et al., 2012). Moreover, cholbut SLN was similarly effective in cell lines whose growth is either sensitive (HCT116) or resistant (HT29) to HDAC inhibitors (Wilson et al., 2010).

Inhibition of cell growth was due to cycle arrest in the G2/M or SubG1 phase and induction of significant cell death.

In our previous work, we showed that cholbut SLN inhibit the ERK and p38 pathways involved in cell growth, migration, and adhesion. Now, we extend those findings by showing that it inhibits the Akt pathway, too, which is overexpressed in a number of cancers, including colon, pancreatic, ovarian, and some steroid hormone-sensitive breast cancers (Roy et al., 2002; Asano et al., 2004). This effect is intriguing since the Akt pathway is critical for prostate cancer invasion (Shukla et al.,  2007), promotes androgen-independent progression, and is essential for neuroendocrine differentiation of the prostate cancer (Li et al., 2007; Wu et al., 2007). Moreover, Akt phosphorylation correlates with cell growth, differentiation, and adhesion (Lai et al., 2010), as well as with the invasion grade, vessel infiltration, metastasis to lymph nodes, and tumor stage in human colon carcinomas (Itoh et al., 2002; Khaleghpour et al., 2004). These data indicate that cholbut SLN have an effect in the signalling machinery of tumor cells, but a wider analysis is needed to better depict these effects.

High concentrations of butyrate (about 2–3 mM) are present in the colon lumen. Since they reach pharmacological effectiveness, colon cancer cells can grow only upon development of escape mechanisms. Among these, a role seems to be played by the decrease of the intracellular concentration of butyrate through downregulation of monocarboxylate transporter 1 (MCT1) and sodium-coupled monocarboxylate transporter 1 (SMCT1) (Davis et al., 2008), which are involved in the transport of butyrate and other SCFA (short chain fatty acids) anions. Therefore, cholbut SLN may overcome this resistance by forcing butyrate uptake in the tumor cell through a transporter-independent mechanism. Indeed, upon treatment with cholbut SLN, substantial uptake of butyrate is already detectable after 15 min, and it then persists inside the cell for several days (Brioschi et al., 2008; Minelli et al., 2012). Development of multidrug resistance in human tumors is one of the main obstacles to the success of cancer chemotherapy. This phenomenon is often associated with increased expression of the mdr1 gene, which encodes P-glycoprotein (Pgp). As an energy-dependent efflux pump, this protein is capable of extruding certain drugs from cells, leading to decreased drug concentrations within the cells and reduced efficacy of drugs (Stein et al., 1996). The human colon carcinoma cell lines HCT116 and HCT15 were selected in this work also because they express the Pgp-mediated type of multidrug resistance intrinsically. However, HCT15 displays a greater multidrug resistance than HCT116 (Iwahshi et al., 1991; Lee et al., 2010), which might explain the higher activity of NaBut on HCT116 than on HCT15. Therefore, the observation that chobut SLN similarly inhibited HC116 and HCT15 supports the hypothesis that they escape the transmembrane efflux pumps responsible for drug elimination.

The in vivo data support the in vitro data and show that cholbut SLN have significant antitumor activity against PC-3 by inhibiting local tumor growth after  s.c. injection of the cell line. Moreover, it inhibited lung metastasis dissemination after i.v. injection of the cell line, which may involve the cholbut SLN effect on tumor cell adhesion, migration, and growth. In the light of these results, cholbut SLN might be used in neoadjuvant chemotherapy, allowing potentially curative surgery, or after surgical resection to inhibit local relapses and metastasis development.

Inflammation is present in most cancer tissues, including those that have no precancerous lesions, and a constant inflammatory state may be necessary to maintain and promote cancer progression to a fully malignant phenotype by supporting tumor remodelling, neoangiogenesis, metastasis dissemination, and even modulation of the anticancer innate immune response from a protective M1-type response to a tumor-promoting M2-type response (Wang et al., 2009). Accordingly, the previously reported anti-inflammatory activity of cholbut SLN (Dianzani et al., 2006), may add value to the anti-cancer activity of this compound since it might help to reset the innate immune response. Since SCID mice display a severe defect of the adaptive immune response but maintain a weak innate immune response (Moser et al., 1993; Thomsen et al., 2008), this anti-inflammatory effect might have had a role in our in vivo experiments.

In conclusion, cholbut SLN seems to be an effective delivery system for cholbut because it decreases the active compound concentration, tumor refractoriness and toxic effects, and expands the compound’s mechanisms of actions, enabling it to act on multiple key fronts of anti-tumor activity, such as tumor cell growth, adhesion, and migration, and recruitment of inflammatory cells.

ACKNOWLEDGEMENTS

This research has been supported by Associazione Italiana Ricerca sul Cancro (AIRC, Milan), Regione Piemonte (Piattaforme Innovative – IMMONC, Converging Technologies NANO-IGT , Fondazione Amici di Jean (Turin), PRIN Project 2009 (MIUR, Roma) and  Progetti di Ricerca di Ateneo San Paolo 2011.Minelli et  al

 

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